The g-protein b subunit interaction domain of ste20p/pak family of protein kinases and uses thereof in bioassays

ABSTRACT

The present invention relates generally to signal transduction through G-protein-coupled receptors and more particularly to the interaction between the β subunit of the heterotrimeric G-protein and the Ste20p/PAK family of protein kinases. More particularly, the invention is directed to the identification of the G-protein β subunit interaction domain of Ste20p/PAK family of protein kinases, the Ste20p/PAK interaction domain of G-protein β subunit, to antibodies specific for these interacting domains, the nucleic acid molecules encoding same, to assays, expression vectors, indicator cells, strains, methods and agents which make use of this Ste20p/PAK—G β  interaction.

FIELD OF THE INVENTION

[0001] The present invention relates generally to signal transduction incells. More particularly, the present invention relates to signaltransduction through G-protein-coupled receptors and especially to theinteraction between β subunits of heterotrimeric G-proteins and theSte20p/PAK family of protein kinases. The invention also relates toassays, expression vectors, strains, methods and agents which make useof this Ste20p/PAK-G_(β) interaction.

BACKGROUND OF THE INVENTION

[0002] The transmission of numerous extracellular signals through thecell membrane, eventually leading to gene expression modulation, iseffected through the interplay of G-protein-coupled receptors (GPCR, oneof the most ubiquitous transmembrane receptor families) and aheterotrimeric complex of nucleotide-binding regulatory proteins. Thiscomplex, also termed tripartite G-proteins or heterotrimeric G-proteins,is comprised of three subunits termed α, β, and γ. These subunits whichcan transduce the extracellular signal through the GPCR downstream todifferent signal transduction pathways are the basis for a wide varietyof cell signalling functions involved for example in intercellularcommunication, response to environmental stimuli such as growth factors,hormones, neurotransmitters, physical parameters (such as light andtemperature) and the like. Of importance, the G-protein dependentsignalling pathway is conserved in organisms ranging from yeast to man.Due to the structural and functional homologies between the G-proteinsin diverse organisms, the yeast Saccharomyces cerevisiae is used as amodel system for higher eukaryotic cells and organisms. In fact,numerous factors involved in G-protein signalling have been shown tofunctionally substitute for the yeast equivalents. The tripartiteG-protein complex for example, was shown to be functionallyreconstituted using mammalian G_(α) and yeast G_(βγ) (WO 95/21925). Inview of the diversity and importance of the signals which induce theG-protein dependent signal transduction pathway, and the importance ofthe downstream effectors of the G-proteins, the dissection of theinteractions taking place in these signal transduction pathways havetremendous fundamental and commercial potential. Furthermore, theseinteractions represent targets for therapeutic agents. Indeed, theimportance of the G-protein-dependent signalling pathway in regulatingcritical cellular biological functions is demonstrated by theidentification of disease conditions which are influenced or determinedby mutations in this pathway. For example, the role of GPCRs in diseaseis reviewed in Coughlin (1994, Curr. Op. Cell. Biol., 6:191-197).Examples of mutations of GPCRs responsible for human diseases have beendescribed (WO 96/41169 and references therein). Moreover, the treatmentof a variety of disease conditions is effected through a modulation ofthe G-protein signalling pathway. For example, agonist analogs ofgonadotropin-releasing hormone have been used to treat breast andprostate cancer, endometriosis and non-tumorous ovarian hyperandrogenicsyndrome (Pace et al., 1992, Am. Fam. Physician, 44:1777-1782). In viewof the critical role played by G-protein signal transduction in cellularhomeostasis and disease conditions there remains a need to identifymodulators of the G-protein signalling pathways downstream from GPCRs.

[0003] The p21-activated protein kinase (PAK) family is a large growingfamily of regulatory enzymes involved in varied cellular processesranging from cellular morphogenesis, stress response and apoptosis. ThePAK family or Ste20p/PAK family was originally identified based on theproperty of its kinases to bind to the activated Rho-type p21GTPasesCdc42 and its related protein Rac1. The signature for this family ofkinases is a characteristic sequence in the subdomain VIII of the kinasedomain (FIG. A; Sells et al., 1997, Trends Cell. Biol., 7:162-167).

[0004] The Ste20p/PAK family of protein kinases is divided into threegroups or sub-families: (1) the so-called true PAKs which contain anN-terminal p21 binding domain (PBD); (2) the pleckstrin-homology (PH)PAKs which also contain a PH-domain upstream of the PBD; and (3) the GCKsub-family exemplified by the germinal center kinase (GCK), which have along C-terminal region and lacking a recognizable PBD (FIG. A).

[0005] Like Raf, PAKs link GTPases to a protein kinase cascade. However,unlike Raf, for which the activation by Ras can be attributed in largepart to a relocalization of the kinase to the plasma membrane, PAK-p21interaction alone is sufficient for in vitro activation. PAK-Rac andRaf-Ras interactions therefore display both common and differentcharacteristics.

[0006] Ste20p kinase, the founding member of the Ste20p/PAK family,shares sequence similarity to protein kinase C, and is required totransmit the pheromone signal from G_(βγ) to downstream components ofthe signalling pathway (Leberer et al., 1992, EMBO J., 11:4815-4824).Ste20p/PAK has been shown to be a pivotal point between theG-protein-coupled receptors/G_(β)-proteins and the mitogen activatedprotein kinase (MAP kinase) pathway (Leberer et al., 1997, Curr.Opinion. Genet. & Devel., 7:59-66).

[0007] The implication of Ste20p in the activation of a protein kinasecascade prompted the analysis of a similar phenomenon in mammaliancells. Although a definite role for Ste20p/PAKs as major effectors inthe stress activated protein kinase cascades (SAPK) has yet to beformally demonstrated, their implication therein has been described(Sells et al., 1997, supra). Indeed, the yeast Ste20p regulated pathwayssuch as mating and filamentous growth share similarities with theJNK/SAPK pathway in mammalian cells which is thought to be activated, atleast in part, by a cascade of small G-proteins and homologs of Ste20p(Leberer et al., 1997, supra). As with Ste20p in yeast, PAKs appear tobe involved in morphological responses such as membrane ruffling and theformation of focal adhesions which might be functionally equivalent tomating protrusions in yeast (Leberer et al., 1997, supra). Further, thesimilarity of Ste20p to mammalian p65 PAK (Leberer et al., 1992, supraand U.S. Pat. No. 5,605,825) and of Cdc42p to the mammalian Rho-likeguanosine triphosphate Rac1, Cdc42Hs and RhoA, which are known toparticipate in the activation of the JNK/SAPK signalling cascade and theregulation of actin reorganization in response to extracellular signals,indicates that signal transduction through Ste20p/PAK may be relevant tothe understanding of similar signalling mechanisms in organisms rangingfrom yeast to mammalian cells (Leeuw et al., 1995, Science,270:1210-1213). The answers obtained using Ste20p in yeast are thereforeof importance in the global understanding of Ste20p/PAK implications invarious signalling cascades in eukaryotes in general.

[0008] Recent examples have shown the importance of theG-protein-coupled receptor-tripartite G-proteins—Ste20p/PAK interactions(Knaus et al., 1995, Science, 269:221-223; Teo et al., 1995, J. Biol.Chem., 270:26690-26697). It has been established that G-protein coupledreceptors can regulate PAKs in mammalian cells. Chemoattractants wereshown to rapidly stimulate two human PAKs through the activation ofheterotrimeric G-proteins leading to the phosphorylation of p47^(phox),suggesting an implication of G-protein-PAKs in NADPH oxidase regulation,and hence, in inflammatory response of human phagocytic leucocytes.Further, thrombin, which binds to a classical G-protein coupled receptorwas shown to activate γ-PAK, a platelet protein kinase displayingsignificant identity to human p65 PAK, suggesting that PAK may be a partof the thrombin-response signalling complex and platelet function (Teoet al., 1995, supra).

[0009] Like PAKs, a number of GCK-like PAK members (referred as group(3) above) activate kinase cascades such as the aforementioned JunN-terminal kinase (JNK) cascade, the stress activated protein kinase(SAPK cascade) and the mitogen activated protein kinase (MAPK cascade).Although sequence similarities between GCK and PAK family members seemlimited primarily to the kinase domain, the identification of the p21binding motifs in the rat homolog of GCK, raises the possibility thatother GCK-PAK-subfamily members might have non-recognized PBDs (Sells etal., 1997, supra).

[0010] The recent identification of HIV's essential protein Nef asassociating with and activating at least one PAK-like kinase furtherindicates that PAKs and homologs thereof have the potential to play animportant role in animal diseases and in human diseases in particular(Sells et al., 1997, supra).

[0011] The mating-pheromone response in yeast provides a geneticallytractable system to study structure/function relationships of theG-protein-Ste20p signal transduction pathway and related pathways invivo. In view of the high degree of functional and structural homologiesbetween the G-proteins and downstream effectors such as the Ste20p/PAKproteins, the yeast system has the potential to provide criticalinsights into signal transduction pathways in higher eukaryotes(Lebereretal., 1992, EMBO J., 11:4805-4813).

[0012] The yeast mating-response MAP kinase cascade consists of Ste11p(a MAP or extracellular signal regulated kinase kinase (MEK) kinasehomolog), Ste7p (a MEK homolog) and the partially redundant MAP kinasehomologs Fus3p and Kss1p (Leberer et al., 1997, supra). Activation ofthis cascade through binding of pheromones to G-protein coupledreceptors induces cellular processes which are typical ofdifferentiating cells, including growth arrest in G₁ of the cell cycle,differential gene expression, and polarized morphogenesis which leads tothe formation of mating-specific projections (Leberer et al., 1997,supra). G_(β)-mediated activation of this cascade involves Ste20p (a MEKkinase kinase) and the MAP kinase scaffolding protein Ste5p (Leberer etal., 1997, supra). PAKs, a subgroup of mammalian Ste20p homologs, can beactivated by either the small G-proteins Cdc42 and Rac or byheterotrimeric G-proteins in various signalling pathways (Sells et al.,1997, supra). The Cdc42p binding domain of Ste20p has been shown to bedispensable for pheromone signalling in yeast suggesting that activationof Ste20p in response to pheromone occurs in a manner independent ofCdc42p (Peter et al., 1996, EMBO J., 15:7046-7059; Leberer et al., 1997,supra).

[0013] The importance of the Ste20p/PAK family of protein kinases issupported by the significant functional and structural conservationthereof throughout evolution. The recent discovery that certain GCK/PAKsubfamily members may also couple with GTPases raises the possibilitythat PAKs in general may mediate GTPase functions. In view of thecritical and often essential roles of such Ste20p/PAK interactions infundamental and diverse cellular processes, and the conservation of thestructure/function relationship of PAKs throughout evolution, there is atremendous need in dissecting and understanding the moleculardeterminants involved in Ste20p/PAK-G-protein interactions. Suchdissections and understandings might shed a light on the possibilitythat differential regulation by heterotrimeric and small G-proteins maycontribute to Ste20p/PAK specificity on the downstream MAP kinasemodule, and may explain how the same protein kinase module may regulatedifferent developmental pathways within the same cell.

[0014] The present invention seeks to meet these and other needs.

[0015] The present description refers to a number of documents, thecontent of which is herein incorporated by reference.

SUMMARY OF THE INVENTION

[0016] The invention concerns the identification of the domainsimplicated in the Ste20p/PAK—Ste4p/G_(β) interaction. More particularly,the invention relates to the G_(β) interaction domain of Ste20p andhomologs thereof.

[0017] The present invention relates to the identification of themolecular determinants of Ste4p/G_(β) interaction in Ste20p/PAK. Theinvention further relates to the identification of a Ste20p/PAKinteraction domain in Ste4p/G_(β).

[0018] Also, the invention relates to a characterization of themolecular determinant of a Ste20p/PAK interaction domain in Ste4p/G_(β).

[0019] The present invention further relates to isolated polypeptidescontaining a Ste4p/G_(β) interaction domain of Ste20p/PAK.

[0020] As well, it relates to isolated polypeptides containing aSte20p/PAK interaction domain of Ste4p/G_(β).

[0021] Further, the invention relates to epitope-binding portions of thepolypeptides of the present invention.

[0022] In a preferred embodiment, the Ste4p/G_(β) interaction domain ofSte20p/PAK comprises the amino acid sequence as set forth in theconsensus sequence SSLφPLI_(V)Xφφβ and as set forth in SEQ. ID. NO.: ID.NO.:27. In a particular embodiment, the Ste20p/G_(β) interaction domainof Ste20p/PAK comprises an amino acid sequence in accordance with theabove consensus sequence. Examples of such sequences include sequencesas set forth in SEQ. ID. NO.: ID. NOs.:1, 2, 4-11 or derivatives orfragments thereof. Ste20p/G_(β) interaction domains having a sequencewith significant homology to the consensus are also provided for examplein SEQ. ID. NO.: ID. NO.:3, 12 and 13 or derivatives or fragmentsthereof.

[0023] In another embodiment, a Ste4p/G_(β) interaction domain ofSte20p/PAK comprises a more divergent amino acid sequence as set forthin SEQ. ID. NO.: ID. NOs.:14-20 or derivatives or fragments thereof, ascompared to the above-listed consensus sequence.

[0024] In yet another preferred embodiment, the Ste20p/PAK interactiondomain of Ste4p/G_(β) comprises the amino acid sequence as set forth inSEQ. ID. NO.: ID. NOs.:21-25 or derivatives or fragments thereof.

[0025] The invention in addition relates to nucleic acid sequencesencoding a Ste4p/G_(β) interaction domain of Ste20p/PAK and to nucleicacid sequences encoding a Ste20p/PAK interaction domain of Ste4p/G_(β).In one particular embodiment, the nucleic acid sequences encoding aSte4p/G_(β) domain of Ste20p/PAK encode the amino acid sequence assetforth in one of SEQ. ID. NO.: ID. NOs.:1-13 or functional derivativesthereof, in SEQ. ID. NO.: ID. NOs.: 14-20 or to a nucleic acid sequencewhich hybridizes thereto under high stringent conditions or is at least90% identical to such nucleic acid sequences encoding the Ste4p/G_(β)binding domain of the present invention.

[0026] In another embodiment, the nucleic acid sequence encoding theSte20p/PAK interaction domain of Ste4p/G_(β) encodes the amino acidsequence as set forth in SEQ. ID. NOs.:21-26 or derivatives or fragmentsthereof or to a nucleic acid sequence which hybridizes thereto underhigh stringent conditions or is at least 90% identical to nucleic acidsequences encoding the Ste20p/PAK interaction domain of the presentinvention. In a preferred embodiment, the nucleic acid sequences of thepresent invention are as set forth in SEQ. ID. NOs.:28 and 30,derivatives or fragments thereof, or nucleic acid sequences whichhybridize thereto under stringent conditions or are at least 90%identical thereto.

[0027] The present invention also seeks to provide a recombinant nucleicacid molecule comprising an isolated nucleic acid of the presentinvention operably linked to a promoter element; cells containing same,and vectors and host cells harboring such vectors for expressing thepolypeptides of the invention.

[0028] The present invention also seeks to provide antibodies directedto the polypeptides or epitope bearing portions thereof as well as tohybridomas producing monoclonal antibodies directed against suchpolypeptides.

[0029] The invention further seeks to provide methods and compositionsto screen for compounds having the ability to modulate a signaltransduction pathway through their modulation of theSte20p/PAK—Ste4p/G_(β) interaction. In one aspect of the presentinvention, the compound inhibits the Ste20p/PAK—Ste4p/G_(β) interactionand uncouples the G-protein receptor from downstream cascades. Inanother aspect, the agent enhances the Ste20p/PAK—Ste4p/G_(β)interaction, thereby inducing the activation of a downstream signaltransduction cascade. In a particular aspect of the present invention,the abilities of a compound(s) to modulate a signal transduction pathwaythrough their modulation of the Ste20p/PAK—Ste4p/G_(β) interaction isassessed by measuring effects on cellular metabolism. In a particularembodiment, this assessment is made through the use of yeast cells asindicator cells and the effect of the test compound(s) observed throughthe mating ability of the yeast cells. In another particular embodiment,this assessment is made through in vitro means well known to the personof ordinary skill. Non limiting examples of such in vitro means includeenzyme-linked immunosorbent assays (ELISA) or other immunologicalassays, filter binding assays, scintillation proximity assays and thelike. Once identified such Ste20p/PAK—Ste4p/G_(β) modulating agents canbe used as lead compounds to search for drugs, that can modulate aparticular signal transduction pathway.

[0030] The present invention is also directed to pharmaceuticalcompositions for controlling diseases which are dependent on theinteraction between Ste20p/PAK and Ste4p/G_(β). As well, the inventionrelates to the administration of such compositions to an animalsuffering from a disease which is dependent on the aforementionedinteraction.

[0031] Accordingly, the present invention also seeks to provide an assaykit for screening and identifying compounds which modulate theSte20p/PAK—Ste4p/G_(β) interaction wherein the kit contains a firstpolypeptide comprising a Ste4p/G_(β) interaction domain of Ste20p/PAKand a second polypeptide comprising a Ste20p/PAK interaction domain ofSte4p/G_(β), and wherein the interaction of the interacting domains isassayable.

[0032] The present invention in addition seeks to provide a method forscreening and identifying compounds which modulate theSte20p/PAK—Ste4p/G_(β) interaction, comprising the step of incubating acompound in admixture with a substantially purified first and secondpolypeptide, wherein the first polypeptide comprises a Ste4p/G_(β)interaction domain of Ste20p/PAK and the second polypeptide comprises aSte20p/PAK interaction domain of Ste4p/G_(β), and determining the extentto which the compound modulates the interaction between the twopolypeptides as compared to a control incubation in the absence of thecompound.

[0033] In a particular aspect, the present invention seeks to provide amethod of controlling diseases, dependent on an interaction ofSte20p/PAK and Ste4p/G_(β) in an animal such as a mammal and topharmaceutical compositions therefor.

[0034] In addition, the present invention seeks to provide a non-humanorganism containing the nucleic acid molecule encoding an interactiondomain of the present invention. The present invention also seeks toprovide a non-human organism containing a knock-out of an interactiondomain of the present invention.

[0035] The polypeptides and nucleic acid sequences of the presentinvention have utility in designing in vitro and in vivo experimentalmodels. Such experimental models enable the screening of largecollections of synthetic, semi-synthetic, or natural compounds fortherapeutic use in Ste20p/PAK—Ste4p/G_(β)-dependent diseases orapplications. The present invention also enables the identification ofsignalling pathways converging at the Ste20p/PAK—G_(β)Ste4p interaction.

[0036] The applicant is the first to demonstrate a direct interactionbetween Ste20p/PAK and G/Ste4p. Before the present invention, it was notclear whether Ste20p PAK and G_(β) interacted. In addition, theapplicant is the first to identify the domains involved in theinteraction of Ste20p/PAK with Ste4p/G_(β), of relevance to theunderstanding of signal transduction in all eukaryotic organisms.

[0037] In accordance with the present invention, there is thereforeprovided polypeptidic regions involved in the interaction of Ste20p/PAKand Ste4p/G_(β). As well there is provided nucleic acid moleculesencoding such interacting domains. Further, there is provided fusionproteins comprising the interaction domains of the present invention,nucleic acid molecules encoding same and cells harboring those nucleicacid molecules.

[0038] In accordance with the present invention, there is also provided,assays and methods for the identification of compounds which modulatethe Ste20p/PAK—Ste4p/G_(β) interaction.

[0039] In accordance with the present invention, there is additionallyprovided methods of treatment and uses of compounds which modulateSte20p/PAK—Ste4p/G_(β) interaction as well as pharmaceuticalcompositions containing same.

[0040] Furthermore, in accordance with the present invention, there isprovided, a composition of matter comprising: an isolatedSte4p/G_(β)-binding polypeptide or fragment thereof wherein the isolatedSte4p/G_(β)-binding polypeptide is a Ste20p/PAK polypeptide whichdirectly binds to a Ste4p/G_(β) polypeptide or fragment thereof; and anisolated Ste20p/PAK-binding polypeptide or fragment thereof, wherein theisolated Ste20p/PAK polypeptide is a Ste4p/G_(β) polypeptide whichdirectly binds to a Ste20p/PAK polypeptide or fragment thereof. Inaddition, in accordance with the present invention, there is alsoprovided, a composition of matter comprising an isolated nucleic acidmolecule comprising a nucleic acid sequence which encodes aSte4p/G_(β)-binding domain of Ste20p/PAK and an isolated nucleic acidmolecule comprising a nucleic acid sequence which encodes aSte20p/PAK-binding domain of Ste4p/G_(β).

[0041] It shall also be understood, that since there is significanthomology between the different members of the Ste20p/PAK family membersand between the evolutionary divergent Ste4p/G_(β) sequences (seebelow), that the person of ordinary skill, will be able to adapt theteachings of the present invention in a variety of ways, with amino acidand nucleic acid sequences from different animals and organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Having thus generally described the invention, reference will nowbe made to the accompanying drawings, showing by way of illustration apreferred embodiment thereof, and in which:

[0043] FIG. A (prior art) shows a structural comparison of the extendedp21-activated Ste20p/PAK family of protein kinases (Sells et al., 1997,Trends in Cell Biol., 7:162-167);

[0044]FIG. 1 shows the association of Ste4p with Ste20p and Ste5p inyeast cells. (A) Time course of pheromone-induced Ste20p binding toHA-Ste4p. HA-Ste4p expressed from the STE4 promoter in cells deleted forSTE4 was immunoprecipitated after treatment with 1 pM a-factor. Relativeamounts of Ste20p and HA-Ste4p were determined in Western blots (seeexample in upper and middle panels) and quantified densitometrically(mean values ±SD, n=3) (lower panel). (B) Association of Ste5p withHA-Ste4p expressed from the STE4 promoter in cells deleted for STE4 .HA-Ste4p immunoprecipitates from exponentially growing (−) andpheromone-treated (90 minutes) (+) yeast cells were analyzed withantibodies to Ste5p (upper panel) or HA-Ste4p (lower panel). (C)Overexpression of Ste4p leads to binding to Ste20p. HA-Ste4p wasoverexpressed from the GAL1 promoter in cells deleted for STE20 (lane 1)or STE4 (lanes 2 and 3). HA-Ste4p expression was suppressed inglucose-containing medium in cells deleted for STE4 (lane 4).Immunoprecipitates obtained with antibodies to Ste20p (lanes 1 and 2) orthe HA-epitope (lanes 3 and 4) were analyzed for the presence of Ste20p(upper panel) and HA-Ste4p (lower panel). (D) Coimmunoprecipitation ofHA-Ste4p and Ste20p truncation mutants. HA-Ste4p and Ste20p⁴⁹⁵⁻⁸⁸⁸(lanes 1 and 3) or Ste20p⁴⁹⁵⁻⁸⁷⁷ (lanes 2 and 4) truncation mutants wereoverexpressed from the GAL1 promoter in cells deleted for STE20.HA-Ste4p (lanes 1 and 2) and Ste20p (lanes 3 and 4) immunoprecipitateswere analyzed for the presence of Ste20p (upper panel) and HA-Ste4p(lower panel) by Western blot analyses. Multiple bands of HA-Ste4p andSte20p represent phosphorylated forms as indicated by phosphatasetreatment (data not shown).

[0045]FIG. 2 shows the In vitro-G_(β) binding assays. (A) Ste4p binds toa sequence carboxyl-terminal to the kinase domain of Ste20p. GST fusionswith full length Ste20p (Leberer et al., 1997, supra) (GST-Ste20p FL)and the indicated Ste20p fragments were incubated with invitro-translated ³⁵S-Ste4p in the presence (left, right and (+) inmiddle panels) or in the absence ((−), middle panel) of invitro-translated HA-Ste18p. GST fusion proteins were detected by Westernblot analyses with antibodies to GST (upper panels). ³⁵S-Ste4p wasdetected by autoradiography (lower panels). The presence of HA-Ste18pwas confirmed by Western blot analyses (data not shown). (B) Summary ofthe interactions between Ste20p fragments and Ste4p. The interactionswere determined by either in vitro binding assays (a) orcoimmunoprecipitations from yeast extracts (b). Conserved residues areunderlined in multiple alignments of carboxyl-terminal sequences ofSte20p (Leberer et al., 1992, supra), mouse mPAK3 (Bagrodia et al.,supra), rat PAK (Manser et al., 1994, Nature, 367:40-46) and yeast Cla4p(Cvrckova et al., 1995, Genes Dev., 9:1817-1830), and human PAK, CBD,Cdc42p binding domain. (C) Interactions of ³⁵S-Ste4p with mouse mPAK3and yeast Cla4p. GST and amino-terminal fusions of GST with Ste20p,mouse mPAK3 and Cla4p were incubated with in vitro-translated ³⁵S-Ste4pin the presence (+) or absence (−) of in vitro-translated HA-Ste18p.Analyses of proteins were performed as described in (A). Relativeamounts of ³⁵S-Ste4p were normalized for relative levels of full lengthGST fusion proteins containing the intact carboxyl-terminal Ste4pbinding site. Data are given as percent of the amount of ³⁵S-Ste4p boundto GST-Ste20p (mean values ±SD, n>3).

[0046]FIG. 3 shows the mutational analyses of the association of Ste4pwith Ste20p. Interaction of Ste4p mutants with Ste20p and Ste5p. Fusionsof GST with Ste20p and Ste5p were incubated in the presence of invitro-translated HA-Ste18p with wild-type ³⁵S-Ste4p or the indicateddominant-negative ³⁵S-Ste4p mutants (Leberer et al., 1992, supra).Relative amounts of the GST fusion proteins and of ³⁵S-Ste4p werequantified by densitometric evaluation of Western blots andautoradiographs, respectively. Data are given as percentage of bindingof wild-type ³⁵S-Ste4p (mean values ±SD, n≧3).

[0047]FIG. 4 shows a model for the role of Ste20p in the activation ofthe pheromone response pathway.

[0048]FIG. 5 shows multiple alignments of the G_(β)-binding sequence ofSte20p with the homologous regions of related protein kinases of theSte20p/PAK family. All accession numbers are from the Swiss Prot and PIRor GeneBank (in parentheses) databases. Numbers to the left of the firstresidue from each sequence indicates the position of this residue in theprotein sequence (where 1 is the initiator Met). Number to the rightdepicts the position of the carboxyl terminal residue. Numbers inparathesis are from incomplete sequences. The consensus sequence for theG_(β)-binding motif is show below (where φ is either A, I, L, M, S, orT, and β is a basic residue). Sc, Saccharomyces cerevisiae; Ca, Candidaalbicans; Sp, Schizosaccharomyces pombe; Hs, Homo sapiens; Dm,Drosophila melanogaster; Xen, Xenopus; Ce, Caenorabditis elegans; Dd,Dictyostelium discoidium; Ac, Acantamoeba.

[0049]FIG. 6 shows multiple alignments of yeast Ste4p with mammalian Gβsubunits (Hgbb1, human Gβ1; Hggb2, human Gβ2; Hggb3, human Gβ3; Mgbb4,mouse Gβ4; Mgbb5, mouse Gβ5). The numbers in parentheses are the SwissProt accession numbers.

[0050] Other objects, advantages and features of the present inventionwill become more apparent upon reading of the following non-restrictivedescription of preferred embodiments with reference to the accompanyingdrawings which are exemplary and should not be interpreted as limitingthe scope of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0051] The domains involved in the Ste20p/PAK—Ste4p/G_(β) interactionare herein provided. Isolated polynucleotides and oligonucleotidesencoding the Ste20p/PAK—Ste4p/G_(β) interaction domains are provided bythe present invention. Isolated proteins encoded by thesepolynucleotides and oligonucleotides are also provided. Examples ofamino acid sequences in accordance with the present invention includeSEQ. ID. Nos.:1-27, 29, and 31. Examples of nucleic acid sequences inaccordance with the present invention and from which fragments andderivatives thereof can be obtained include SEQ. ID. Nos.:28 and 30.

[0052] Certain aspects of the present invention also include nucleicacid sequences which are homologous to the nucleic acid sequences of thepresent invention.

[0053] In another embodiment of the invention, the amino acid sequencesof the present invention provide sequences for obtaining polyclonal ormonoclonal antibodies, chimeric antibodies, humanized antibodies and thelike which are specific for the Ste20p/PAK—Ste4p/G_(β) interactiondomain.

[0054] Alternatively, in another embodiment the present inventionprovides a simple, rapid high-throughput functional bioassay foridentifying compounds that modulate the Ste20p/PAK—Ste4p/G_(β)interaction. These compounds can act either as agonists or antagonistsof Ste20p/PAK—Ste4p/G_(β) interaction and signalling functions. In oneembodiment, the assay is an “in vivo” experimental model based on theincubation of indicator cells with test compounds and the identificationof the test compound as agonist or antagonist of Ste20p/PAK—Ste4p/G_(β)interaction. Alternatively, it is based on the use of an “in vitro”experimental model such as an enzymatic assay, binding assay and thelike (i.e. examples 8 and 9). Compounds can be tested individually or inpools or libraries. The term “antagonist” refers to a compound whichinhibits the interaction between Ste20p/PAK and Ste4p/G_(β), therebyuncoupling signal transduction through G-proteins. Alternatively, theterm “agonist” refers to a compound that stimulates such a signaltransduction by promoting Ste20p/PAK—Ste4p/G_(β) interaction. The term“modulator” is used herein to refer to a compound or a mixture or poolthereof which positively or negatively affect the Ste20p/PAK—Ste4p/G_(β)interaction.

[0055] As used herein, the terms “interaction domains” and “bindingdomains” are used interchangeably.

[0056] As used herein the recitation “indicator cells” refers to cellsthat express an interaction domain of a Ste4p/G_(β)—Ste20p/PAK and aSte20p/PAK interaction domain of Ste4p/G_(β), and wherein an interactionbetween these domains is coupled to an identifiable or selectablephenotype or characteristic such that it provides an assessment of theinteraction between the domains. Such indicator cells can be used in thescreening assays of the present invention. In a preferred embodiment,the indicator cells have been engineered so as to replace at least oneof the endogenous Ste20p/PAK and Ste4p/G_(β) interacting domains ofSte4p/G_(β) and Ste20p/PAK respectively, by a chosen derivative,fragment, homolog, or mutant thereof. Alternatively, the indicator cellsare engineered so as to inhibit the expression of at least one of theaforementioned endogenous interacting domains. The cells can be yeastcells or higher eukaryotic cells such as mammalian cells (WO 96141169).Preferably, the indicator cells are yeast cells. Non-limiting examplesof such cells and vectors are exemplified herein below (i.e. examples 7and 11). In one particular embodiment, an indicator cell of the presentinvention which is wild type with respect to mating can be used to testa compound or a library thereof in order to identify same which affectmating. In another embodiment, the indicator cell can be a yeast cellharboring vectors enabling the use of the two hybrid system technologyas well known in the art (Ausubel et al. 1994, supra). In oneembodiment, a reporter gene encoding selectable marker can be operablylinked to a control element such that expression of the selectablemarker is dependent on the interaction of the Ste20p/PAK—Ste4p/G_(β)interacting domains. Such an indicator cell could be used to rapidlyscreen at high-throughput a vast array of test compounds. In aparticular embodiment, the reporter gene is luciferase, β-Gal or greenfluorescent protein. It will be understood that the indicator cell,polypeptides and nucleic acids of the present invention can beengineered to be particularly suited for the expression of heterologousSte20p/PAK and/or Ste4p/G_(β) proteins (WO 95/21925).

[0057] As exemplified herein below in one embodiment, at least one of aSte20p/PAK and Ste4p/G_(β) interaction domain of the present inventionmay be provided as a fusion protein. The design of constructs thereforand the expression and production of fusion protein are well known inthe art (Sambrook et al., 1989, in Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratories, and Ausubel et al., 1994,Current Protocols in Molecular Biology, Wiley, N.Y.). In certainembodiments, it might be beneficial to introduce a protease cleavagesite between the two polypeptide sequences which have been fused.Non-limiting examples of such fusion proteins include ahemagglutinin—Ste4p/G_(β) fusion protein and a Ste20p-GST fusion. Incertain embodiments, it might be beneficial to fuse the interactiondomains of the present invention to signal peptide sequences enabling asecretion of the fusion protein from the host cell. Signal peptides fromdiverse organisms are well known in the art. Bacterial OmpA and yeastSuc2 are two non-limiting examples of proteins containing signalsequences.

[0058] As used herein, the term “compound” is used broadly to refer tonatural, synthetic or semi-synthetic compounds. The term “compound”therefore denotes for examples macromolecules, cell or tissue extracts(from plants or animals). Non-limiting examples of compounds includenucleic acid molecules, peptides, antibodies, carbohydrates andpharmaceutical agents. The agents can be selected and screened by avariety of means including random screening, rational selection and byrational design using for example protein or ligand modelling methodssuch as computer modelling. The terms “rationally selected” or“rationally designed” are meant to define compounds which have beenchosen based on the configuration of the interaction domains of thepresent invention. As will be understood by the person of ordinaryskill, macromolecules having non-naturally occurring modifications arealso within the scope of the term “compound”. For example,peptidomimetics, well known in the pharmaceutical industry and generallyreferred to as peptide analogs can be generated by modelling asmentioned above. Similarly, in a preferred embodiment, the polypeptidesof the present invention are modified to enhance their stability. Itshould be understood that in most cases this modification should notalter the biological activity of the interaction domain. The compoundsidentified in accordance with the teachings of the present inventionhave a therapeutic value for the treatment of diseases or conditionswhich are dependent on Ste20p/PAK—Ste4p/G_(β) interaction. Such diseasesor conditions could include proliferative diseases, inflammatorydiseases, apoptosis and the like.

[0059] As used herein, the term “selectable marker” is used broadly torefer to markers which confer an identifiable trait to the indicatorcell. Non-limiting example of selectable markers include markersaffecting viability, metabolism, proliferation, morphology and the like.

[0060] As used herein, agonists and antagonists ofSte20p/PAK—Ste4p/G_(β) interaction also include potentiators of knowncompounds with such agonist or antagonist properties. In one embodiment,agonists can be detected by contacting the indicator cell with acompound or mixture or library of compounds for a fixed period of time.The level of gene expression (e.g. the level of luciferase produced)within the treated cells is then determined. The expression level can becompared to that of the reporter gene in the absence of the compound(s).The difference between the levels of gene expression indicates whetherthe compound(s) of interest agonize the aforementioned interaction. Themagnitude of the level of reporter gene product expressed (treated vs.untreated cells) provides a relative indication of the strength of thatcompound(s) as an agonist. Alternatively, such an indicator cell can beused to identify antagonists.

[0061] For example, the test compound or compounds are incubated withthe host cell in conjunction with one or more known agonists held at afixed concentration. An indication and relative strength of theantagonistic properties of the compound(s) can be provided by comparingthe level of gene expression in the indicator cell in the presence ofthe known agonist, in the absence of test compounds vs in the presencethereof.

[0062] It shall be understood that the “in vivo” experimental model canalso be used to carry out an “in vitro” assay. For example, cellularextracts from the indicator cells can be prepared and used in one of theaforementioned “in vitro” tests (i.e. example 11). Numerous in vitromethods to detect and/or quantify the interaction between twointeracting polypeptides are known to the person of ordinary skill. Forexample, antibodies can be used for this purpose. The conditions and thetype of assay can be adapted by the person of ordinary skill as afunction of the desired type of information required, the format of theassay, the detection method and the type and nature of the antibodyused. Non limiting examples of commonly known immunological assays whichcan be used to assess the interaction between Ste20p/PAK and Ste4p/G_(β)include radioimmunoassays, ELISA, immunofluorescence-type assays and thelike. Immunological assays which can be used in the context of thepresent invention are described for example in Harlow et al., 1988 (in:Antibody—A Laboratory Manual, CSH Laboratories). As well different typeof binding assays, for example direct or indirect, or competitivebinding assays can be used. Scintillation proximity-type assays areother non limiting examples of assays which can be used to identifycompounds which modulate the Ste20p/PAK—Ste4p/G_(β) interaction.

[0063] For certainty, as used herein “Ste20p/PAK” and “Ste4p/G_(β)”refer herein to members of the Ste20p/PAK family of protein kinases andto homologs of “G_(β)”, respectively. Thus, any Ste20p/PAK or anySte4p/G_(β) family member with the proviso that it comprises theinteraction domains of the present invention or nucleic acid sequencesencoding same can be used to practice the present invention. Forcertainty, the sequences and polypeptides useful to practice theinvention include without being limited thereto mutants, homologs,subtypes, alleles and the like. It shall be understood that generally,the sequences of the present invention should encode a functional(albeit defective) interaction domain. It will be clear to the person ofordinary skill that whether an interaction domain of the presentinvention, variant, derivative, or fragment thereof retains its functionin binding to its partner can be readily determined by using theteachings and assays of the present invention and the general teachingsof the art. As exemplified herein below, the interaction domains of thepresent invention can be modified, for example by in vitro mutagenesis,to dissect the structure-function relationship thereof and permit abetter design and identification of modulating compounds. However, somederivative or analogs having lost their biological function ofinteracting with their respective interaction partner (Ste20p/PAK orSte4p/G_(β)) may still find utility, for example for raising antibodies.Such analogs or derivatives could be used for example to raiseantibodies to the interaction domains of the present invention. Theseantibodies could be used for detection or purification purposes. Inaddition, these antibodies could also act as competitive ornon-competitive inhibitor and be found to be modulators ofSte20p/PAK—Ste4p/G_(β) interaction.

[0064] A consensus sequence of the Ste4p/G_(β) interaction domain isherein provided. It shall be clear that a 100% identity to thisconsensus sequence is not necessary to provide functionality toSte20p/PAK (binding to Ste4p/G_(β)) since for example (and as describedbelow), a serine to alanine substitution at the first aa positionthereof (DPak; SEQ. ID. NO.: ID. NO.:12) retains the biologicalfunction. The same can be said of SEQ. ID. NO.: ID. NO.:3, since Shk1 ofS.Pombe complements a Ste20 gene disruption. More divergent amino acidsequences, as exemplified for example by SEQ. ID. NO.: ID. NO.:17 doesnot bind, however. Thus, more divergent amino acid sequence such as SEQ.ID. NO.: ID. NOs.:14-20 and especially SEQ. ID. NO.: ID. NOs.:17-20 canbe used to identify compounds and/or molecular determinants of thesequence which can stimulate the Ste4p/G_(β) interaction.

[0065] Nucleotide sequences are presented herein by single strand, inthe 5′ to 3′ direction, from left to right, using the one letternucleotide symbols as commonly used in the art and in accordance withthe recommendations of the IUPAC-IUB Biochemical NomenclatureCommission.

[0066] The present description refers to a number of routinely usedrecombinant DNA (rDNA) technology terms. Nevertheless, definitions ofselected examples of such rDNA terms are provided for clarity andconsistency.

[0067] As used herein, “isolated nucleic acid molecule”, refers to apolymer of nucleotides. Non-limiting examples thereof include DNA andRNA molecules purified from their natural environment.

[0068] The term “recombinant DNA” as known in the art refers to a DNAmolecule resulting from the joining of DNA segments. This is oftenreferred to as genetic engineering.

[0069] The term “DNA segment”, is used herein, to refer to a DNAmolecule comprising a linear stretch or sequence of nucleotides. Thissequence when read in accordance with the genetic code, can encode alinear stretch or sequence of amino acids which can be referred to as apolypeptide, protein, protein fragment and the like.

[0070] The nucleic acid (i.e. DNA or RNA) for practicing the presentinvention may be obtained according to well known methods.

[0071] A host cell or indicator cell has been “transfected” by exogenousor heterologous DNA (e.g. a DNA construct) when such DNA has beenintroduced inside the cell. The transfecting DNA may or may not beintegrated (covalently linked) into the genome of the cell. Inprokaryotes, yeast, and mammalian cells for example, the transfectingDNA may be maintained on an episome such as a plasmid. With respect toeukaryotic cells, a stably transfected cell is one in which thetransfecting DNA has become integrated into the genome so that it isinherited by daughter cells upon replication. The stability of theintegrated DNA can be demonstrated by the establishment of cell lines orclones comprised of a population of daughter cells containing thetransfecting DNA. Transfection methods are well known in the art(Sambrook et al., 1989, supra; Ausubel et al., 1994, supra).

[0072] “Nucleic acid hybridization” refers generally to thehybridization of two single-stranded nucleic acid molecules havingcomplementary base sequences, which under appropriate conditions willform a thermodynamically favored double-stranded structure. Examples ofhybridization conditions can be found in the two laboratory manualsreferred above (Sambrook et al., 1989, supra, and Ausubel et al., 1994,supra) and are commonly known in the art. In the case of a hybridizationto a nitrocellulose filter, as for example in the well known Southernblotting procedure, a nitrocellulose filter can be incubated overnightat 65° C. with a labeled probe in a solution containing 50% formamide,high salt (5× SSC or 5× SSPE), 5× Denhardt's solution, 1% SDS, and 100μg/ml denatured carrier DNA (i.e. salmon sperm DNA). Thenon-specifically binding probe can then be washed off the filter byseveral washes in 0.2× SSC/0.1% SDS at a temperature which is selectedin view of the desired stringency: room temperature (low stringency),42° C. (moderate stringency) or 65° C. (high stringency). The selectedtemperature is based on the melting temperature (Tm) of the DNA hybrid.Of course, RNA-DNA hybrids can also be formed and detected. In suchcases, the conditions of hybridization and washing can be adaptedaccording to well known methods by the person of ordinary skill.Stringent conditions will be preferably used (Sambrook et al.,1989,supra).

[0073] As used herein, the term “gene” is well known in the art andrelates to a nucleic acid sequence defining a single protein orpolypeptide. A “structural gene” defines a DNA sequence which istranscribed into RNA and translated into a protein having a specificamino acid sequence thereby giving rise the a specific polypeptide orprotein.

[0074] A “heterologous” (i.e. a heterologous gene) region of a DNAmolecule is a subsegment segment of DNA within a larger segment that isnot found in association therewith in nature. The term “heterologous”can be similarly used to define two polypeptidic segments not joinedtogether in nature. Non-limiting examples of heterologous genes includereporter genes such as luciferase, chloramphenicol acetyl transferase,β-galactosidase, and the like which can be juxtaposed or joined toheterologous control regions or to heterologous polypeptides.

[0075] The term “vector” is commonly known in the art and defines aplasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNAvehicle into which DNA of the present invention can be cloned. Numeroustypes of vectors exist and are well known in the art.

[0076] The term “expression” defines the process by which a structuralgene is transcribed into mRNA (transcription), the mRNA is then beingtranslated (translation) into one polypeptide (or protein) or more.

[0077] The terminology “expression vector” defines a vector or vehicleas described above but designed to enable the expression of an insertedsequence following transformation into a host. The cloned gene (insertedsequence) is usually placed under the control of control elementsequences such as promoter sequences. The placing of a cloned gene undersuch control sequences is often referred to as being operably linked tocontrol elements or sequences.

[0078] Expression control sequences will vary depending on whether thevector is designed to express the operably linked gene in a prokaryoticor eukaryotic host or both (shuttle vectors) and can additionallycontain transcriptional elements such as enhancer elements, terminationsequences, tissue-specificity elements, and/or translational initiationand termination sites.

[0079] As used herein, the designation “functional derivative” denotes,in the context of a functional derivative of a sequence whether annucleic acid or amino acid sequence, a molecule that retains abiological activity (either function or structural) that issubstantially similar to that of the original sequence. This functionalderivative or equivalent may be a natural derivatives or may be preparedsynthetically. Such derivatives include amino acid sequences havingsubstitutions, deletions, or additions of one or more amino acids,provided that the biological activity of the protein is conserved. Thesame applies to derivatives of nucleic acid sequences which can havesubstitutions, deletions, or additions of one or more nucleotides,provided that the biological activity of the sequence is generallymaintained. When relating to a protein sequence, the substituting aminoacid as chemico-physical properties which are similar to that of thesubstituted amino acid. The similar chemico-physical properties include,similarities in charge, bulkiness, hydrophobicity, hydrophylicity andthe like. The term “functional derivatives” is intended to include“fragments”, “segments”, “variants”, “analogs” or “chemical derivatives”of the subject matter of the present invention.

[0080] Thus, the term “variant” refers herein to a protein or nucleicacid molecule which is substantially similar in structure and biologicalactivity to the protein or nucleic acid of the present invention.

[0081] The functional derivatives of the present invention can besynthesized chemically or produced through recombinant DNA technology.all these methods are well known in the art. In view of the conservationof the Ste4p/G_(β) binding domain of Ste20p/PAK throughout evolution(see below), it will be apparent to the person of ordinary skill thatsequences from different organisms and animals and chimeras thereof canbe used in accordance with the teachings of the present invention.

[0082] As used herein, “chemical derivatives” is meant to coveradditional chemical moieties not normally part of the subject matter ofthe invention. Such moieties could affect the physico-chemicalcharacteristic of the derivative (i.e. solubility, absorption, half lifeand the like, decrease of toxicity). Such moieties are exemplified inRemington's Pharmaceutical Sciences (1980). Methods of coupling thesechemical-physical moieties to a polypeptide are well known in the art.

[0083] The term “allele” defines an alternative form of a gene whichoccupies a given locus on a chromosome.

[0084] As commonly known, a “mutation” is a detectable change in thegenetic material which can be transmitted to a daughter cell. As wellknown, a mutation can be, for example, a detectable change in one ormore deoxyribonucleotide. For example, nucleotides can be added,deleted, substituted for, inverted, or transposed to a new position.Spontaneous mutations and experimentally induced mutations exist. Theresult of a mutations of nucleic acid molecule is a mutant nucleic acidmolecule. A mutant polypeptide can be encoded from this mutant nucleicacid molecule.

[0085] As used herein, the term “purified” refers to a molecule havingbeen separated from a cellular component. Thus, for example, a “purifiedprotein” has been purified to a level not found in nature. A“substantially pure” molecule is a molecule that is lacking in all othercellular components.

[0086] In general, techniques for preparing antibodies (includingmonoclonal antibodies and hybridomas) and for detecting antigens usingantibodies are well known in the art (Campbell, 1984, In “MonoclonalAntibody Technology: Laboratory Techniques in Biochemistry and MolecularBiology”, Elsevier Science Publisher, Amsterdam, The Netherlands) and inHarlow et al., 1988 (supra). The present invention also providespolyclonal, monoclonal antibodies, or humanized versions thereof,chimeric antibodies and the like which inhibit or neutralize theirrespective interaction domains and/or are specific thereto.

[0087] The term “non-human animals” refers to animals having atransgenic interruption or alteration of an endogenous gene encoding aninteraction domain of the present invention (knock-out animal) and/oranimals having an interruption into the genome in which a transgene(directing the expression of encoding an interaction domain of, or thepresent invention) has been introduced. Non-limiting examples of suchnon-human animals include vertebrates such as rodents, non-humanprimates, amphibians, reptiles and the like. These animals are preparedin accordance with known methods.

[0088] The present invention also relates to a kit for identifyingcompounds which modulate Ste 20p/PAK—Ste4p/G_(β) interaction, comprisinga nucleic acid, a protein or a ligand in accordance with the presentinvention. For example, a compartmentalized kit in accordance with thepresent invention includes any kit in which reagents are contained inseparate containers. Such containers include small glass containers,plastic containers or strips of plastic or paper. Such containers allowthe efficient transfer of reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers could include a container which will accept the testcompound, a container which contains the interacting domains used in theassay, containers which contain enzymes, containers which contain washreagents, and containers which contain the reagents used to detect theextent of interaction of the interacting domains.

[0089] The present invention is described in further detail in thefollowing non-limiting examples.

EXAMPLE 1

[0090] Yeast Strains and Manipulations

[0091]S. cerevisiae strains used herein were W303-1A (MA Ta ade2 leu2trp1 ura3 his3 can1), YEL206 (W303-1A ste20Δ-3::TRP1) (Wu et al., 1995,J. Biol. Chem., 270:15984-15992), YEL155 (W303-1A ste5Δ::TRP1) andYEL121 (W303-1A ste4Δ::LEU2). Mating assays, analysis of matingprojection formation, and growth arrest, induction of FUSI::lacZ andcomplementation assays of the growth defect of cells deleted for bothSTE20 and CLA4 were carried out as described (Leberer et al., 1997,supra; Leberer et al., 1993, Genet., 241:241-254).

EXAMPLE 2

[0092] Construction of Plasmids

[0093] To construct pBTL110 carrying HA-STE4P/GB under control of theSTE4P promoter, a fragment from nucleotides −491 to −1 of STE4P wasamplified by PCR and cloned into pRS313 (Sikorski et al., 1989,Genetics, 122:19-27). The BamHI fragment of pL55 (Whiteway et al., 1995,Science, 269:1572-1575) was then subcloned downstream of the STE4P/GBpromoter.

[0094] To create pBTL38 and pBTL65 carrying STE4P/GB and HA-STE18 undercontrol of the T3 RNA polymerase promoter, STE4P/GB and HA-STE18 wereamplified by PCR and ligated into pRS316 and pRS313 (Sikorski et al.,1989, supra), respectively.

[0095] To create pBTL79, pBTL80, pBTL81 and pBTL82 carrying theSTE4^(D62N), STE4^(K55E), STE4^(N157H/S175P) and STE4^(ΔF177) mutantsunder control of the T7 RNA polymerase promoter, respectively, the GAL1promoter was excised from pGAL-STE4P/GB-D62N, pGAL-STE4P/GB-K55E,pGAL-STE4P/GB-N157H/S175P and pGAL-STE4P/GB-ΔF177, respectively (Lebereret al., 1992, supra).

[0096] To create pDH171 and pDH172 carrying the Ste20p⁴⁹⁵⁻⁸⁷⁷ andSte20p⁴⁹⁵⁻⁸⁸⁸ fragments under control of the GAL1 promoter,respectively, these fragments were amplified by PCR and subcloned intopRS313GAL (Leberer et al., 1992, supra).

[0097] To create pBTL83, pBTL84, pBTL146 and pBTL147 carrying fusions ofGST with the Ste20p⁸⁷⁶⁻⁹³⁹, Ste20p⁸⁷⁶⁻⁸⁹², Ste20p⁸¹⁹⁻⁸⁷⁵ andSte20p⁸¹⁹⁻⁸⁹² fragments, respectively, fragments were amplified by PCRand subcloned into pGEX-4T-1 (Pharmacia).

[0098] To create a fusion of GST with full length Ste5p, the STE5 codingregion was amplified by PCR and ligated into pGEX-4T-3 (Pharmacia) toyield pVL50.

EXAMPLE 3

[0099] Oligodeoxynucleotide-directed Mutagenesis of STE20

[0100] pBTL151 and pBTL150 carrying the STE20 mutants STE20^(CLA4), inwhich the sequence encoding amino acids 879 to 887 of STE20 was replacedby the sequence of CLA4 encoding amino acids 832 to 840 (Cvrckova etal., 1995, supra), and STE20^(S879A/S880A/P883A), respectively, undercontrol of the STE20 promoter, were created by site directed mutagenesis(Kunkel et al., 1987, Methods in Enzymology, 154:367-382). The mutationswere confirmed by sequencing. To create pBTL117 and pBTL118 carryingfusions of GST with the fragments from amino acid 819 to 939 of theSTE20^(S879A/S880A/P883A) and STE20^(CLA4) mutants, respectively, thesefragments were amplified by PCR and subcloned into pGEX-4T-2(Pharmacia).

EXAMPLE 4

[0101] Immunochemical Procedures

[0102] Immunoprecipitation experiments with specific antibodies to theHA-epitope (12CA5 monoclonal and rabbit polyclonal anti-HA antibodieswere from Babco, Richmond), Ste20p (Wu et al., 1995, J. Biol. Chem.270:15984) and Ste5p (Wall et al., 1995, Cell 83:1047-1058) wereperformed according to standard procedures as described (Whiteway etal., 1995, supra; Leeuw et al., 1995, supra). For the detection ofSte20p fragments, a secondary sheep antibody specific to rabbitimmunoglobulin light chains and a tertiary HRP-conjugated donkeyantibody to sheep IgG were obtained from The Binding Site, Lim.Immunoprecipitations were confirmed in at least three independentexperiments. For quantitation, immunoblots were evaluated by integratingdensitometry using an Epson ES 1200-C densitometer and the NIH Image1.59 software.

EXAMPLE 5

[0103] In vitro G_(β) Binding Assays

[0104] Plasmids were linearized downstream of the termination codons ofthe respective genes. In vitro-transcription was performed by usingeither T3 or T7 RNA polymerase and m⁷G(5′)ppp(5′)G capped GTP. Invitro-translation of the resulting mRNA was carried out with ³⁵S-abeledmethionine using an in vitro-translation kit (Promega).

[0105] GST fusion proteins were purified on glutathione-Sepharose beadsin 20 mM HEPES buffer pH 7.4, containing 100 mM NaCl, 50 mM NaF, 0.5 MSorbitol, 2 mM EDTA, 1 mM Na₃VO₄, 0.1% Triton X-100, 1% BSA (wt/v) and aprotease inhibitor cocktail, and washed 5 times in phosphate bufferedsaline (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH7.4) by centrifugation at 1.000 g. Proteins (5-10 μg) were incubatedwith 5 μl of the reticulocyte lysate containing the in vitro-translatedproducts in 25 μl of PBS for 10 minutes at 30° C.

[0106] The beads were then washed three times with PBS, separated bySDS-PAGE and analysed by autoradiography of Western blots. Results wereconfirmed in at least three independent experiments. Immunodetection andevaluation of immunoblots and radiographs were then performed asdescribed above. Data obtained for ³⁵S-Ste4p were corrected for relativeconcentrations of the respective GST fusion proteins.

EXAMPLE 6

[0107] In vivo Association of Ste20p with G_(β) (Ste4p)

[0108] Coimmunoprecipitation experiments were performed to analyze thein vivo-association of Ste20p with an influenza hemagglutinin(HA)-epitope tagged version of G_(β) (HA-Ste4p). Antibodies to HA-Ste4pprecipitated low amounts of Ste20p (FIG. 1A). An approximately 5-foldincrease in the interaction between Ste4p and Ste20p was observedalready after 3 minutes of pheromone treatment and maintained for up to15 minutes of stimulation (FIG. 1A). The initial induction ofSte20p/Ste4p complexes is consistent with the time course described forthe stimulation of Far1p (a cyclin inhibitor) and the MAP kinases Fus3pand Kss1p (Chang et al., 1992, Mol. Biol. Cell., 3:445-450; Gartner etal., 1992, Genes Dev., 6:1280-1292) and may be required to activate theMAP kinase cascade for the induction of growth arrest andtranscriptional activation. Additional formation of complexes afterprolonged treatment with pheromone (FIG. 1A) followed a time courseconcomitant with the formation of mating projections and accumulation ofreceptors, Ste20p and Ste4p (Leberer et al., 1997, Curr. Op. Genet. &Dev., 7:59-66; Leberer et al., 1997, EMBO J., 16:83-97) in the tips ofmating projections, and could be involved in the control ofmorphological changes that may require Ste20p dependent phosphorylationof myosin-l or activation of the PKC pathway (Leberer et al., 1997, EMBOJ., 16:83-97; Wu et al., 1996, J. Biol. Chem., 271:31787-31790).

[0109] When the pheromone response pathway was activated throughoverexpression of HA-Ste4p, Ste5p also formed a complex with Ste4p(Whiteway et al., 1995, supra). This complex was present in cellswithout an activated pathway when HA-Ste4p was expressed at wild-typelevels (FIG. 1B), and the association was not significantly alteredafter treatment of cells with pheromone (FIG. 1B), suggesting aconstitutive interaction between Ste4p and Ste5p. Constitutiveactivation of the pheromone signaling pathway through overexpression ofHA-Ste4p stimulated the association of Ste4p with Ste20p in the absenceof pheromone (FIG. 1C). This association required the function ofSte18p, the γ-subunit of the mating response G-protein, but did notrequire the presence of Ste5p (data not shown).

EXAMPLE 7

[0110] Identification of the G_(β) Interaction Domain of Ste20p

[0111] Cells overexpressing the Ste20p⁴⁹⁵⁻⁸⁸⁸ fragment were normal intheir mating functions, whereas cells overexpressing the Ste20p⁴⁹⁵⁻⁸⁷⁷fragment were defective (Table 1). Briefly, Strain YEL206 deleted forSTE20 was transformed with pDH 166 (Whiteway et al., 1995, supra),pDH171 and pDH172 carrying either wild-type STE20 (STE20^(WT)) or theSTE20⁴⁹⁵⁻⁸⁷⁷ and STE20⁴⁹⁵⁻⁸⁸⁸ mutant alleles, respectively, undercontrol of the GAL1 promoter. Mating efficiencies represent mean values±SD (n=3). Mating functions were analyzed as described (Leberer et al.,1997, supra; Leberer et al., 1993, supra). TABLE 1 Effects ofcarboxy-terminal truncations on signaling functions of Ste20p MatingFUS1:: lacZ STE20 efficiencies expression Shmoo allele (%) Basal InducedG₁ arrest formation STE20^(WT) 89.8 ± 15.5  0.1 221.2 + + STE20⁴⁹⁵⁻⁸⁸⁸75.2 ± 7.5  1.6 147.7 + + STE20⁴⁹⁵⁻⁸⁷⁷ 0.02 ± 0.015 <0.1 <0.1 − −

[0112] These results suggest that the region from amino acids 877 to 888carboxyl-terminal to the kinase domain of Ste20p plays an important rolein the pheromone response. This region was also required forcoimmunoprecipitation with HA-Ste4p (FIG. 1D), underlining thephysiological importance of the association between Ste4p and Ste20p inpheromone signaling.

EXAMPLE 8

[0113] In vitro Association of Ste20p with G_(β) (Ste4p)

[0114] [³⁵S]methonine-labeled Ste4p³⁵ (S-Ste4p) and HA-Ste18p weresynthesized in an in vitro-translation system and analyzed for theirability to bind to fusions of glutathione S-transferase (GST) withwild-type Ste20p and fragments of Ste20p (FIG. 2A). It was found that afragment carboxyl-terminal to the kinase domain encompassing residues876 to 892 was necessary and sufficient to bind ³⁵S-Ste4p (FIG. 2A). Thebinding did not depend on the presence of HA-Ste18p (FIGS. 2A,C), andHA-Ste18p alone was not able to bind Ste20p (data not shown). Assummarized in FIG. 2B, these results, together with data obtained in theimmunoprecipitation experiments, suggest that the non-catalytic regionfrom amino acids 876 to 888 of Ste20p represents a binding site forG_(β).

EXAMPLE 9

[0115] The G_(β) Interaction Domain of Ste20p is Functionally Conservedin Mouse mPAK3 and in Ste20p/PAK Members

[0116] Consistent with observations that mammalian PAK isoforms cancomplement the mating defect of yeast cells deleted for STE20 (Bagrodiaet al., 1995, J. Biol. Chem., 270:22731-22737) and that the G_(β)bindingsite is conserved in these kinases (FIG. 2B), mouse mPAK3 (Bagrodia etal., 1995, supra) bound ³⁵S-Ste4p (FIG. 2C).

EXAMPLE 10

[0117] Identification of the Molecular Determinants of the G_(β)Interaction Domain of Ste20p/PAK—

[0118] Ste20p and its closely related isoform Cla4p share a redundantfunction that is essential for cellular viability in yeast (Cvrckova etal., 1995, supra). Consistent with observations that only high levels ofCla4p after overexpression partly complement the mating defect of yeastcells deleted for STE20 (data not shown) and the Ste4p binding site ofSte20p is not well conserved in Cla4p (FIG. 2B), only weak binding ofSte4p to Cla4p was observed (FIG. 2C). These results support the viewthat residues conserved in the Ste4p binding sites of Ste20p and PAKisoforms (FIG. 2B) contribute to the binding of G_(β). These resultsalso provide a weak consensus sequence for G_(β) binding, the sequenceof the G_(β) binding domain of Cla4p. This weak G_(β) binding consensussequence can be used in assays to identify compounds which can stimulateCla4p-G_(β) interaction. In a particular embodiment, the assay involvesthe use of agents to identify agonists of the Cla4p-G_(β) interactionthat will enable a complementation of the mating defect of yeast cellsdeleted for Ste20p. In another embodiment, the physical interactionbetween Cla4p and G_(β) could be assessed in vitro through gel shifts,immunoprecipitation and the like, as well known to the person ofordinary skill and as shown herein. Further, single mutations (or acombination of mutations) in the G-binding domain of Cla4p couldidentify the minimal primary structure requirements enabling adequateG_(β)-Cla4p binding and perhaps the complementation of the Ste20p nullmutant strain mentioned above. A very similar approach is exemplifiedwith single mutations and a triple mutations of the conserved residuesof Ste20p (see below).

EXAMPLE 11

[0119] In vitro Mutagenesis of the G_(β) Interaction Domain of aSte20p/PAK Member

[0120] Single alterations of the conserved residues S879, S880 or P883to alanine did not affect the in vivo-function of Ste20p (data notshown). However, the triple mutantSte₂₀p^(S879A/S880A/P883A in which the highly conserved sequence motif SSLxPL was altered to AALxAL, showed strong defects in mating functions (Table)2). Briefly, for GST (control) and fusions of GST with thecarboxyl-terminal fragments from amino acids 819 to 939 of wild-typeSte20p (STE20^(WT)), the Ste₂₀p^(S879A/S880A/P883A) mutant and theSte20^(CLA4) mutant (in which the sequence encoding amino acids 879 to887 of STE20 was replaced by the sequence of CLA4 encoding amino acids832 to 840; Cvrckova et al., 1995, supra), respectively, were incubatedwith in vitro-translated ³⁵S-Ste4p in the presence of invitro-translated HA-Ste18p. Data are given as relative levels of bound³⁵S-Ste4p normalized against binding to wild-type Ste20p⁸¹⁹⁻⁹³⁹. Forcells deleted for STE20 were transformed with pRS313 (control), pSTE20-5carrying wild-type STE20 (Leberer et al., 1992, supra) (STE20^(WT)),pBTL150 carrying the STE20^(S879A/S880A/P883A) mutant, and pBTL151carrying the STE20^(CLA4) mutant. Proteins were expressed under controlof the STE20 promoter. Finally in vitro-kinase activities weredetermined in immune complexes isolated from YEL206 cells expressing theindicated STE20 alleles or the inactive STE20^(K649R) mutant (Wu et al.,1995, supra) as a control. In vitro-kinase assays were performed asdescribed (Wu et al., 1995, supra) with myelin basic protein (MBP) assubstrate. Data are given as percentage of MBP phosphorylation bywild-type Ste20p. Similar defects were also observed for the mutantSte20p^(CLA4), in which the Ste4p binding site of Ste20p was replaced bythe equivalent region of Cla4p (Table 2 and FIG. 2B). No differenceswere found for the in vitro-kinase activities of these mutants whencompared with wild-type Ste20p (Table 2), and the mutants were found tocomplement the growth defect of cells deleted for both STE20 and CLA4(data not shown). However, binding to ³⁵S-Ste4p was strongly reducedwhen fusions of GST with both mutant versions were analyzed in the invitro-binding assay (Table 2). Thus, the mating defects of these Ste20pmutants correlated with their reduced ability to bind Ste4p. TABLE 2Effect of carboxy-terminal mutations in Ste20p on in vitro-binding toSte4p, in vivo-signaling functions and in vitro-kinase activitiesBinding to Mating Kinase ³⁵S-Ste4p efficiencies FUS1:: lacZ G₁ Shmooactivity STE20 allele (%)^(a)) (%)^(b)) expression^(b)) arrest^(b))formation^(b)) (%)^(c)) STE20^(WT) 100 72.7 ± 6.8  274 ± 41  + + 100STE20^(S879A/S880A/P883A) 15.2 ± 7.8 0.039 ± 0.002  14 ± 1.8 − −  104 ±11.2 STE20^(CLA4) 11.1 ± 9.3 0.016 ± 0.001  11 ± 1.4 − −   93 ± 10.7Control  2.1 ± 1.8 <0.005 <0.2 ± 1   − − 2.5 ± 1.3

EXAMPLE 12

[0121] Identification of the Ste20p Binding Domain of Ste4p

[0122] Mutations within two regions of Ste4p which, when overexpressed,inhibited the signaling function of the wild-type protein werepreviously identified (Leberer et al., 1992, supra). The effect of twoof these dominant-negative mutations within each region were examinedfor their effect on the association of Ste4p with either Ste20p orSte5p. The K55E and D62N mutants of Ste4p (Leberer et al., 1992, supra)were defective in binding to GST-Ste20p, whereas binding to GST-Ste5pwas normal (FIG. 3). The inability of these Ste4p mutants to bind Ste20pcorrelated with their sterile phenotype (Leberer et al., 1992, supra).However, the N157H/S175P and ΔF177 mutants which were also found topossess reduced signaling functions (Leberer et al., 1992, supra) wereable to bind both Ste20p and Ste5p, although binding of Ste5p wasreduced when compared with binding to wild-type Ste4p (FIG. 3),suggesting that this region may be involved in the interaction with anas yet unidentified component. The present invention therefore furtherprovides means to identify this unidentified component and a furtherdissection of the structure-function relationship of Ste20p/PAK insignalling function.

[0123] Modeling of Ste4p by using the crystal structure of mammalianG_(β1) (Wall et al., 1995, Cell 83:1047-1058) as a template indicatesthat the residues predicted to interact with Ste20p are part of anamino-terminal a-helix in the region of G_(β) that interacts with G_(γ)(Wall et al., 1995, supra; Sondek et al., 1996, Nature, 379:369-374).The structure of yeast Ste4p (G_(β)) was modelled to the structure ofmammalian G_(β1) (Wall et al., supra) using the homology module ofInsight (Biosym, Inc.). Insertions specific for Ste4p were notconsidered.

[0124] However, consistent with the finding that the Ste4p^(D62N) mutantinteracted normally with Ste18p in the two-hybrid system (data notshown), the side chains of these residues are not predicted to beinvolved in the interaction with G_(γ) but rather to be exposed on thecytoplasmic face of the G_(β) structure (data not shown).

[0125] Conclusion

[0126] Together, these results indicate that transmission of thepheromone signal involves the regulated interaction between themating-response G-protein β-subunit and a conserved sequence in theSte20p protein kinase (FIG. 4). Pheromone-induced interaction with Ste4pmay bring Ste20p in vicinity of Ste11p (FIG. 4) which interacts withSte5p (Leberer et al., 1997, Curr. Op. Genet. & Dev., 7:59-66) and canserve as an in vitro-substrate for Ste20p (Wu et al., 1995, J. Biol.Chem., supra). Low concentrations of Ste20p/G_(β) complexes present inthe absence of pheromone may account for the basal signalling levelsfound in uninduced cells and may guarantee the rapid responsiveness ofcells to pheromone (Chang et al., 1992, supra; Gartner et al., 1992,supra). In view of the high degree of conservation of Ste20p familyprotein kinases (Sells et al, 1997, supra), the results presented hereinsuggest that the interaction of these kinases with the β-subunit ofheterotrimeric G-proteins (which are also highly conserved) maycontribute to linking Ste20p homologs to G-protein-coupled receptors inother organisms including mammalian cells.

[0127] Although the present invention has been described hereinabove byway of preferred embodiments thereof, it can be modified, withoutdeparting from the spirit and nature of the subject invention as definedin the appended claims.

1 31 1 423 PRT Unknown Organism Description of Unknown Organism unknown1 Met Ala Ala His Gln Met Asp Ser Ile Thr Tyr Ser Asn Asn Val Thr 1 5 1015 Gln Gln Tyr Ile Gln Pro Gln Ser Leu Gln Asp Ile Ser Ala Val Glu 20 2530 Asp Glu Ile Gln Asn Lys Ile Glu Ala Ala Arg Gln Glu Ser Lys Gln 35 4045 Leu His Ala Gln Ile Asn Lys Ala Lys His Lys Ile Gln Asp Ala Ser 50 5560 Leu Phe Gln Met Ala Asn Lys Val Thr Ser Leu Thr Lys Asn Lys Ile 65 7075 80 Asn Leu Lys Pro Asn Ile Val Leu Lys Gly His Asn Asn Lys Ile Ser 8590 95 Asp Phe Arg Trp Ser Arg Asp Ser Lys Arg Ile Leu Ser Ala Ser Gln100 105 110 Asp Gly Phe Met Leu Ile Trp Asp Ser Ala Ser Gly Leu Lys GlnAsn 115 120 125 Ala Ile Pro Leu Asp Ser Gln Trp Val Leu Ser Cys Ala IleSer Pro 130 135 140 Ser Ser Thr Leu Val Ala Ser Ala Gly Leu Asn Asn AsnCys Thr Ile 145 150 155 160 Tyr Arg Val Ser Lys Glu Asn Arg Val Ala GlnAsn Val Ala Ser Ile 165 170 175 Phe Lys Gly His Thr Cys Tyr Ile Ser AspIle Glu Phe Thr Asp Asn 180 185 190 Ala His Ile Leu Thr Ala Ser Gly AspMet Thr Cys Ala Leu Trp Asp 195 200 205 Ile Pro Lys Ala Lys Arg Val ArgGlu Tyr Ser Asp His Leu Gly Asp 210 215 220 Val Leu Ala Leu Ala Ile ProGlu Glu Pro Asn Ser Glu Asn Ser Ser 225 230 235 240 Asn Thr Phe Ala SerCys Gly Ser Asp Gly Tyr Thr Tyr Ile Trp Asp 245 250 255 Ser Arg Ser ProSer Ala Val Gln Ser Phe Tyr Val Asn Asp Ser Asp 260 265 270 Ile Asn AlaLeu Arg Phe Phe Lys Asp Gly Met Ser Ile Val Ala Gly 275 280 285 Ser AspAsn Gly Ala Ile Asn Met Tyr Asp Leu Arg Ser Asp Cys Ser 290 295 300 IleAla Thr Phe Ser Leu Phe Arg Gly Tyr Glu Glu Arg Thr Pro Thr 305 310 315320 Pro Thr Tyr Met Ala Ala Asn Met Glu Tyr Asn Thr Ala Gln Ser Pro 325330 335 Gln Thr Leu Lys Ser Thr Ser Ser Ser Tyr Leu Asp Asn Gln Gly Val340 345 350 Val Ser Leu Asp Phe Ser Ala Ser Gly Arg Leu Met Tyr Ser CysTyr 355 360 365 Thr Asp Ile Gly Cys Val Val Trp Asp Val Leu Lys Gly GluIle Val 370 375 380 Gly Lys Leu Glu Gly His Gly Gly Arg Val Thr Gly ValArg Ser Ser 385 390 395 400 Pro Asp Gly Leu Ala Val Cys Thr Gly Ser TrpAsp Ser Thr Met Lys 405 410 415 Ile Trp Ser Pro Gly Tyr Gln 420 2 340PRT Unknown Organism Description of Unknown Organism unknown 2 Met SerGlu Leu Asp Gln Leu Arg Gln Glu Ala Glu Gln Leu Lys Asn 1 5 10 15 GlnIle Arg Asp Ala Arg Lys Ala Cys Ala Asp Ala Thr Leu Ser Gln 20 25 30 IleThr Asn Asn Ile Asp Pro Val Gly Arg Ile Gln Met Arg Thr Arg 35 40 45 ArgThr Leu Arg Gly His Leu Ala Lys Ile Tyr Ala Met His Trp Gly 50 55 60 ThrAsp Ser Arg Leu Leu Val Ser Ala Ser Gln Asp Gly Lys Leu Ile 65 70 75 80Ile Trp Asp Ser Tyr Thr Thr Asn Lys Val His Ala Ile Pro Leu Arg 85 90 95Ser Ser Trp Val Met Thr Cys Ala Tyr Ala Pro Ser Gly Asn Tyr Val 100 105110 Ala Cys Gly Gly Leu Asp Asn Ile Cys Ser Ile Tyr Asn Leu Lys Thr 115120 125 Arg Glu Gly Asn Val Arg Val Ser Arg Glu Leu Ala Gly His Thr Gly130 135 140 Tyr Leu Ser Cys Cys Arg Phe Leu Asp Asp Asn Gln Ile Val ThrSer 145 150 155 160 Ser Gly Asp Thr Thr Cys Ala Leu Trp Asp Ile Glu ThrGly Gln Gln 165 170 175 Thr Thr Thr Phe Thr Gly His Thr Gly Asp Val MetSer Leu Ser Leu 180 185 190 Ala Pro Asp Thr Arg Leu Phe Val Ser Gly AlaCys Asp Ala Ser Ala 195 200 205 Lys Leu Trp Asp Val Arg Glu Gly Met CysArg Gln Thr Phe Thr Gly 210 215 220 His Glu Ser Asp Ile Asn Ala Ile CysPhe Phe Pro Asn Gly Asn Ala 225 230 235 240 Phe Ala Thr Gly Ser Asp AspAla Thr Cys Arg Leu Phe Asp Leu Arg 245 250 255 Ala Asp Gln Glu Leu MetThr Tyr Ser His Asp Asn Ile Ile Cys Gly 260 265 270 Ile Thr Ser Val SerPhe Ser Lys Ser Gly Arg Leu Leu Leu Ala Gly 275 280 285 Tyr Asp Asp PheAsn Cys Asn Val Trp Asp Ala Leu Lys Ala Asp Arg 290 295 300 Ala Gly ValLeu Ala Gly His Asp Asn Arg Val Ser Cys Leu Gly Val 305 310 315 320 ThrAsp Asp Gly Met Ala Val Ala Thr Gly Ser Trp Asp Ser Phe Leu 325 330 335Lys Ile Trp Asn 340 3 340 PRT Unknown Organism Description of UnknownOrganism unknown 3 Met Ser Glu Leu Glu Gln Leu Arg Gln Glu Ala Glu GlnLeu Arg Asn 1 5 10 15 Gln Ile Arg Asp Ala Arg Lys Ala Cys Gly Asp SerThr Leu Thr Gln 20 25 30 Ile Thr Ala Gly Leu Asp Pro Val Gly Arg Ile GlnMet Arg Thr Arg 35 40 45 Arg Thr Leu Arg Gly His Leu Ala Lys Ile Tyr AlaMet His Trp Gly 50 55 60 Thr Asp Ser Arg Leu Leu Val Ser Ala Ser Gln AspGly Lys Leu Ile 65 70 75 80 Ile Trp Asp Ser Tyr Thr Thr Asn Lys Val HisAla Ile Pro Leu Arg 85 90 95 Ser Ser Trp Val Met Thr Cys Ala Tyr Ala ProSer Gly Asn Phe Val 100 105 110 Ala Cys Gly Gly Leu Asp Asn Ile Cys SerIle Tyr Ser Leu Lys Thr 115 120 125 Arg Glu Gly Asn Val Arg Val Ser ArgGlu Leu Pro Gly His Thr Gly 130 135 140 Tyr Leu Ser Cys Cys Arg Phe LeuAsp Asp Asn Gln Ile Ile Thr Ser 145 150 155 160 Ser Gly Asp Thr Thr CysAla Leu Trp Asp Ile Glu Thr Gly Gln Gln 165 170 175 Thr Val Gly Phe AlaGly His Ser Gly Asp Val Met Ser Leu Ser Leu 180 185 190 Ala Pro Asp GlyArg Thr Phe Val Ser Gly Ala Cys Asp Ala Ser Ile 195 200 205 Lys Leu TrpAsp Val Arg Asp Ser Met Cys Arg Gln Thr Phe Ile Gly 210 215 220 His GluSer Asp Ile Asn Ala Val Ala Phe Phe Pro Asn Gly Tyr Ala 225 230 235 240Phe Thr Thr Gly Ser Asp Asp Ala Thr Cys Arg Leu Phe Asp Leu Arg 245 250255 Ala Asp Gln Glu Leu Leu Met Tyr Ser His Asp Asn Ile Ile Cys Gly 260265 270 Ile Thr Ser Val Ala Phe Ser Arg Ser Gly Arg Leu Leu Leu Ala Gly275 280 285 Tyr Asp Asp Phe Asn Cys Asn Ile Trp Asp Ala Met Lys Gly AspArg 290 295 300 Ala Gly Val Leu Ala Gly His Asp Asn Arg Val Ser Cys LeuGly Val 305 310 315 320 Thr Asp Asp Gly Met Ala Val Ala Thr Gly Ser TrpAsp Ser Phe Leu 325 330 335 Lys Ile Trp Asn 340 4 340 PRT UnknownOrganism Description of Unknown Organism unknown 4 Met Gly Glu Met GluGln Leu Arg Gln Glu Ala Glu Gln Leu Lys Lys 1 5 10 15 Gln Ile Ala AspAla Arg Lys Ala Cys Ala Asp Val Thr Leu Ala Glu 20 25 30 Leu Val Ser GlyLeu Glu Val Val Gly Arg Val Gln Met Arg Thr Arg 35 40 45 Arg Thr Leu ArgGly His Leu Ala Lys Ile Tyr Ala Met His Trp Ala 50 55 60 Thr Asp Ser LysLeu Leu Val Ser Ala Ser Gln Asp Gly Lys Leu Ile 65 70 75 80 Val Trp AspSer Tyr Thr Thr Asn Lys Val His Ala Ile Pro Leu Arg 85 90 95 Ser Ser TrpVal Met Thr Cys Ala Tyr Ala Pro Ser Gly Asn Phe Val 100 105 110 Ala CysGly Gly Leu Asp Asn Met Cys Ser Ile Tyr Asn Leu Lys Ser 115 120 125 ArgGlu Gly Asn Val Lys Val Ser Arg Glu Leu Ser Ala His Thr Gly 130 135 140Tyr Leu Ser Cys Cys Arg Phe Leu Asp Asp Asn Asn Ile Val Thr Ser 145 150155 160 Ser Gly Asp Thr Thr Cys Ala Leu Trp Asp Ile Glu Thr Gly Gln Gln165 170 175 Lys Thr Val Phe Val Gly His Thr Gly Asp Cys Met Ser Leu AlaVal 180 185 190 Ser Pro Asp Phe Asn Leu Phe Ile Ser Gly Ala Cys Asp AlaSer Ala 195 200 205 Lys Leu Trp Asp Val Arg Glu Gly Thr Cys Arg Gln ThrPhe Thr Gly 210 215 220 His Glu Ser Asp Ile Asn Ala Ile Cys Phe Phe ProAsn Gly Glu Ala 225 230 235 240 Ile Cys Thr Gly Ser Asp Asp Ala Ser CysArg Leu Phe Asp Leu Arg 245 250 255 Ala Asp Gln Glu Leu Ile Cys Phe SerHis Glu Ser Ile Ile Cys Gly 260 265 270 Ile Thr Ser Val Ala Phe Ser LeuSer Gly Arg Leu Leu Phe Ala Gly 275 280 285 Tyr Asp Asp Phe Asn Cys AsnVal Trp Asp Ser Met Lys Ser Glu Arg 290 295 300 Val Gly Ile Leu Ser GlyHis Asp Asn Arg Val Ser Cys Leu Gly Val 305 310 315 320 Thr Ala Asp GlyMet Ala Val Ala Thr Gly Ser Trp Asp Ser Phe Leu 325 330 335 Lys Ile TrpAsn 340 5 340 PRT Unknown Organism Description of Unknown Organismunknown 5 Met Ser Glu Leu Glu Gln Leu Arg Gln Glu Ala Glu Gln Leu ArgAsn 1 5 10 15 Gln Ile Gln Asp Ala Arg Lys Ala Cys Asn Asp Ala Thr LeuVal Gln 20 25 30 Ile Thr Ser Asn Met Asp Ser Val Gly Arg Ile Gln Met ArgThr Arg 35 40 45 Arg Thr Leu Arg Gly His Leu Ala Lys Ile Tyr Ala Met HisTrp Gly 50 55 60 Tyr Asp Ser Arg Leu Leu Val Ser Ala Ser Gln Asp Gly LysLeu Ile 65 70 75 80 Ile Trp Asp Ser Tyr Thr Thr Asn Lys Met His Ala IlePro Leu Arg 85 90 95 Ser Ser Trp Val Met Thr Cys Ala Tyr Ala Pro Ser GlyAsn Tyr Val 100 105 110 Ala Cys Gly Gly Leu Asp Asn Ile Cys Ser Ile TyrAsn Leu Lys Thr 115 120 125 Arg Glu Gly Asp Val Arg Val Ser Arg Glu LeuAla Gly His Thr Gly 130 135 140 Tyr Leu Ser Cys Cys Arg Phe Leu Asp AspGly Gln Ile Ile Thr Ser 145 150 155 160 Ser Gly Asp Thr Thr Cys Ala LeuTrp Asp Ile Glu Thr Gly Gln Gln 165 170 175 Thr Thr Thr Phe Thr Gly HisSer Gly Asp Val Met Ser Leu Ser Leu 180 185 190 Ser Pro Asp Leu Lys ThrPhe Val Ser Gly Ala Cys Asp Ala Ser Ser 195 200 205 Lys Leu Trp Asp IleArg Asp Gly Met Cys Arg Gln Ser Phe Thr Gly 210 215 220 His Ile Ser AspIle Asn Ala Val Ser Phe Phe Pro Ser Gly Tyr Ala 225 230 235 240 Phe AlaThr Gly Ser Asp Asp Ala Thr Cys Arg Leu Phe Asp Leu Arg 245 250 255 AlaAsp Gln Glu Leu Leu Leu Tyr Ser His Asp Asn Ile Ile Cys Gly 260 265 270Ile Thr Ser Val Ala Phe Ser Lys Ser Gly Arg Leu Leu Leu Ala Gly 275 280285 Tyr Asp Asp Phe Asn Cys Ser Val Trp Asp Ala Leu Lys Gly Gly Arg 290295 300 Ser Gly Val Leu Ala Gly His Asp Asn Arg Val Ser Cys Leu Gly Val305 310 315 320 Thr Asp Asp Gly Met Ala Val Ala Thr Gly Ser Trp Asp SerPhe Leu 325 330 335 Arg Ile Trp Asn 340 6 353 PRT Unknown OrganismDescription of Unknown Organism unknown 6 Met Ala Thr Asp Gly Leu HisGlu Asn Glu Thr Leu Ala Ser Leu Lys 1 5 10 15 Ser Glu Ala Glu Ser LeuLys Gly Lys Leu Glu Glu Glu Arg Ala Lys 20 25 30 Leu His Asp Val Glu LeuHis Gln Val Ala Glu Arg Val Glu Ala Leu 35 40 45 Gly Gln Phe Val Met LysThr Arg Arg Thr Leu Lys Gly His Gly Asn 50 55 60 Lys Val Leu Cys Met AspTrp Cys Lys Asp Lys Arg Arg Ile Val Ser 65 70 75 80 Ser Ser Gln Asp GlyLys Val Ile Val Trp Asp Ser Phe Thr Thr Asn 85 90 95 Lys Glu His Ala ValThr Met Pro Cys Thr Trp Val Met Ala Cys Ala 100 105 110 Tyr Ala Pro SerGly Cys Ala Ile Ala Cys Gly Gly Leu Asp Asn Lys 115 120 125 Cys Ser ValTyr Pro Leu Thr Phe Asp Lys Asn Glu Asn Met Ala Ala 130 135 140 Lys LysLys Ser Val Ala Met His Thr Asn Tyr Leu Ser Ala Cys Ser 145 150 155 160Phe Thr Asn Ser Asp Met Gln Ile Leu Thr Ala Ser Gly Asp Gly Thr 165 170175 Cys Ala Leu Trp Asp Val Glu Ser Gly Gln Leu Leu Gln Ser Phe His 180185 190 Gly His Gly Ala Asp Val Leu Cys Leu Asp Leu Ala Pro Ser Glu Thr195 200 205 Gly Asn Thr Phe Val Ser Gly Gly Cys Asp Lys Lys Ala Met ValTrp 210 215 220 Asp Met Arg Ser Gly Gln Cys Val Gln Ala Phe Glu Thr HisGlu Ser 225 230 235 240 Asp Val Asn Ser Val Arg Tyr Tyr Pro Ser Gly AspAla Phe Ala Ser 245 250 255 Gly Ser Asp Asp Ala Thr Cys Arg Leu Tyr AspLeu Arg Ala Asp Arg 260 265 270 Glu Val Ala Ile Tyr Ser Lys Glu Ser IleIle Phe Gly Ala Ser Ser 275 280 285 Val Asp Phe Ser Leu Ser Gly Arg LeuLeu Phe Ala Gly Tyr Asn Asp 290 295 300 Tyr Thr Ile Asn Val Trp Asp ValLeu Lys Gly Ser Arg Val Ser Ile 305 310 315 320 Leu Phe Gly His Glu AsnArg Val Ser Thr Leu Arg Val Ser Pro Asp 325 330 335 Gly Thr Ala Phe CysSer Gly Ser Trp Asp His Thr Leu Arg Val Trp 340 345 350 Ala 7 25 PRTUnknown Organism Description of Unknown Organism unknown 7 Glu Ala AsnSer Ser Leu Ala Pro Leu Val Lys Leu Ala Arg Leu Lys 1 5 10 15 Lys ValAla Glu Asn Met Asp Ala Asp 20 25 8 22 PRT Unknown Organism Descriptionof Unknown Organism unknown 8 Asp Asp Val Ser Ser Leu Ser Pro Leu ValLys Ile Ala Arg Leu Lys 1 5 10 15 Lys Met Ser Glu Ser Asp 20 9 18 PRTUnknown Organism Description of Unknown Organism unknown 9 Val Pro ValSer Ser Leu Ile Pro Leu Ile Lys Ser Ile His His Ser 1 5 10 15 Gly Lys 1021 PRT Unknown Organism Description of Unknown Organism unknown 10 LysPro Leu Ser Ser Leu Thr Pro Leu Ile Ala Ala Ala Lys Glu Ala 1 5 10 15Thr Lys Asn Asn His 20 11 21 PRT Unknown Organism Description of UnknownOrganism unknown 11 Lys Pro Leu Ser Ser Leu Thr Pro Leu Ile Met Ala AlaLys Glu Ala 1 5 10 15 Met Lys Ser Asn Arg 20 12 21 PRT Unknown OrganismDescription of Unknown Organism unknown 12 Lys Pro Leu Ser Ser Leu ThrPro Leu Ile Met Ala Ala Lys Glu Ala 1 5 10 15 Met Lys Ser Asn Arg 20 1321 PRT Unknown Organism Description of Unknown Organism unknown 13 LysPro Leu Ser Ser Leu Thr Pro Leu Ile Ala Ala Ala Lys Glu Ala 1 5 10 15Thr Lys Asn Asn His 20 14 22 PRT Unknown Organism Description of UnknownOrganism unknown 14 Lys Pro Leu Ser Ser Leu Thr Pro Leu Ile Leu Ala AlaLys Glu Ala 1 5 10 15 Ile Lys Asn Ser Ser Arg 20 15 21 PRT UnknownOrganism Description of Unknown Organism unknown 15 Lys Pro Leu Ser SerLeu Thr Pro Leu Ile Leu Ala Ala Lys Glu Ala 1 5 10 15 Met Lys Ser AsnArg 20 16 21 PRT Unknown Organism Description of Unknown Organismunknown 16 Lys Pro Leu Ser Ser Leu Thr Pro Leu Ile Met Ala Ala Lys GluAla 1 5 10 15 Met Lys Ser Asn Arg 20 17 22 PRT Unknown OrganismDescription of Unknown Organism unknown 17 Lys Pro Leu Ser Ser Leu ThrPro Leu Ile Ile Ala Ala Lys Glu Ala 1 5 10 15 Ile Lys Asn Ser Ser Arg 2018 20 PRT Unknown Organism Description of Unknown Organism unknown 18Arg Pro Leu Ala Ser Leu Thr Pro Leu Ile Met Ala Ala Lys Glu Ala 1 5 1015 Thr Lys Gly Asn 20 19 21 PRT Unknown Organism Description of UnknownOrganism unknown 19 Lys Pro Leu Ser Ser Leu Thr Pro Tyr Ile Ile Thr GlyLys Gln Ile 1 5 10 15 Ala Lys Gly Gly His 20 20 23 PRT Unknown OrganismDescription of Unknown Organism unknown 20 Lys Pro Leu Ala Ser Leu TyrTyr Leu Ile Val Ala Ala Lys Lys Ser 1 5 10 15 Ile Ala Glu Ala Ser AsnSer 20 21 26 PRT Unknown Organism Description of Unknown Organismunknown 21 Cys Asn Cys Asn Gly Leu Val Pro Ala Ile Met Glu Ala Lys LysAla 1 5 10 15 Lys Glu Ala His Ser Lys Phe Ser Ile His 20 25 22 25 PRTUnknown Organism Description of Unknown Organism unknown 22 Gly Pro GluSer Asp Leu Ile Pro Leu Val Glu Arg Thr Lys Asn Glu 1 5 10 15 Ala GlnArg Asp Phe Ser Met Phe Phe 20 25 23 14 PRT Unknown Organism Descriptionof Unknown Organism unknown 23 Cys Asp Pro Lys Asp Leu Thr Ser Leu LeuGlu Trp Lys Glu 1 5 10 24 32 PRT Unknown Organism Description of UnknownOrganism unknown 24 Gly Lys Ile Glu Glu Leu Ala Pro Leu Leu Glu Trp LysLys Gln Gln 1 5 10 15 Gln Lys His Gln Gln His Lys Gln Glu Thr Ser AspThr Gly Phe Ala 20 25 30 25 13 PRT Unknown Organism Description ofUnknown Organism unknown 25 Cys Ser Pro Glu Gln Leu Lys Val Ser Leu LysTrp His 1 5 10 26 20 PRT Unknown Organism Description of UnknownOrganism unknown 26 Cys Pro Thr Glu Asp Leu Lys Ser Ile Ile Phe Ser ArgLys Ala Asn 1 5 10 15 Thr His Ile Asn 20 27 11 PRT Artificial SequenceDescription of Artificial Sequence consensus sequence 27 Ser Ser Leu XaaPro Leu Xaa Xaa Xaa Xaa Xaa 1 5 10 28 4136 DNA Saccharomyces cerevisiaeCDS (276)..(3092) 28 gaattcgaaa gtctaccgct tttggcagct gaaaaattcagaaagtcacc tggccagaga 60 ggaaaaatac gaaaccaaaa agaggcatcc gtaaattcgcattagcaacg catgcttaca 120 tagatactca catactacac acacttacat actttcttaaagacatacat ccgtacgtac 180 aattagagcg aggtagcaag caacccaaac ttcttcccttcactgcctca caccccatcc 240 taaatatccc acaagatcct cgactaatac aagaa atg agcaat gat cca tct 293 Met Ser Asn Asp Pro Ser 1 5 gct gta tcg gaa cta ccagac aag gac agt ctt gat aac ggt atc agc 341 Ala Val Ser Glu Leu Pro AspLys Asp Ser Leu Asp Asn Gly Ile Ser 10 15 20 aat gac aat gaa agg gcc atgggc ggc aat ggc gat ggc ggc gat gga 389 Asn Asp Asn Glu Arg Ala Met GlyGly Asn Gly Asp Gly Gly Asp Gly 25 30 35 tta cga tta cca agg acc act ggaact ttg aac gtc aat gcc tta caa 437 Leu Arg Leu Pro Arg Thr Thr Gly ThrLeu Asn Val Asn Ala Leu Gln 40 45 50 aaa ggc act aat gct gcc cat gaa gctggt gga tac aaa tcc atg gat 485 Lys Gly Thr Asn Ala Ala His Glu Ala GlyGly Tyr Lys Ser Met Asp 55 60 65 70 cct gcg aag aac gcg gag aca acc aatgat gat gac aat aat gtc gtt 533 Pro Ala Lys Asn Ala Glu Thr Thr Asn AspAsp Asp Asn Asn Val Val 75 80 85 tca cta gat gat cct att caa ttt acc cgagta tct tcc tcc tct gtc 581 Ser Leu Asp Asp Pro Ile Gln Phe Thr Arg ValSer Ser Ser Ser Val 90 95 100 atc agt gga atg tct tca tcc atg agt cctcat tct aac atc gat gaa 629 Ile Ser Gly Met Ser Ser Ser Met Ser Pro HisSer Asn Ile Asp Glu 105 110 115 acc aaa tct cta gaa gca gtc act cca aacata aat acc agc aat ata 677 Thr Lys Ser Leu Glu Ala Val Thr Pro Asn IleAsn Thr Ser Asn Ile 120 125 130 acc ccg gat cat tct gct gac aac aca ttttct acc ata aat gcg tcc 725 Thr Pro Asp His Ser Ala Asp Asn Thr Phe SerThr Ile Asn Ala Ser 135 140 145 150 gag tca gat cac cag ttt aat gac acttta cta tca aaa ctg tcg tta 773 Glu Ser Asp His Gln Phe Asn Asp Thr LeuLeu Ser Lys Leu Ser Leu 155 160 165 aca gat tct aca gaa act ata gaa aataac gcg aca gtg aag cac cag 821 Thr Asp Ser Thr Glu Thr Ile Glu Asn AsnAla Thr Val Lys His Gln 170 175 180 cag cca gtt gca tct tcc aca gta aactcg aat aag agc tcc act gat 869 Gln Pro Val Ala Ser Ser Thr Val Asn SerAsn Lys Ser Ser Thr Asp 185 190 195 ata cga agg gct aca cca gtg tcc actccc gtt atc tct aaa cca tcg 917 Ile Arg Arg Ala Thr Pro Val Ser Thr ProVal Ile Ser Lys Pro Ser 200 205 210 atg aca acc acg cca aga cag atc aattca gct tcc cat tcg ctt tcg 965 Met Thr Thr Thr Pro Arg Gln Ile Asn SerAla Ser His Ser Leu Ser 215 220 225 230 aac cct aag cat aag caa cat aaacca aaa gtt aaa ccg tcc aag cct 1013 Asn Pro Lys His Lys Gln His Lys ProLys Val Lys Pro Ser Lys Pro 235 240 245 gaa gca aaa agt aaa ccg gtt tctgtg aaa aaa agc ttt cct tcg aaa 1061 Glu Ala Lys Ser Lys Pro Val Ser ValLys Lys Ser Phe Pro Ser Lys 250 255 260 aat cct tta aaa aac tcc tct ccacct aaa aag caa aca gaa aaa tcg 1109 Asn Pro Leu Lys Asn Ser Ser Pro ProLys Lys Gln Thr Glu Lys Ser 265 270 275 tat tat tct tcc tct tcg aaa aaaagg aaa agc ggt tca aat agt ggt 1157 Tyr Tyr Ser Ser Ser Ser Lys Lys ArgLys Ser Gly Ser Asn Ser Gly 280 285 290 aca cta aga atg aaa gat gtc tttacg tcc ttt gta cag aat ata aag 1205 Thr Leu Arg Met Lys Asp Val Phe ThrSer Phe Val Gln Asn Ile Lys 295 300 305 310 aga aat tct cag gat gat aaaagg gcc tca tcg tcg tcc aat aat tct 1253 Arg Asn Ser Gln Asp Asp Lys ArgAla Ser Ser Ser Ser Asn Asn Ser 315 320 325 tcc tca tct tct ata acc accgct ttg agg ata tct acg cca tac aat 1301 Ser Ser Ser Ser Ile Thr Thr AlaLeu Arg Ile Ser Thr Pro Tyr Asn 330 335 340 gcc aag cat atc cac cat gtgggc gtg gac tcc aag act ggt gag tac 1349 Ala Lys His Ile His His Val GlyVal Asp Ser Lys Thr Gly Glu Tyr 345 350 355 aca ggt ttg ccg gag gaa tgggaa aaa ttg ttg act tct agt ggt att 1397 Thr Gly Leu Pro Glu Glu Trp GluLys Leu Leu Thr Ser Ser Gly Ile 360 365 370 tcc aaa aga gaa caa cag caaaac atg caa gca gtc atg gat att gtc 1445 Ser Lys Arg Glu Gln Gln Gln AsnMet Gln Ala Val Met Asp Ile Val 375 380 385 390 aaa ttc tat cag gat gtcacg gaa aca aac ggt gaa gat aaa atg ttc 1493 Lys Phe Tyr Gln Asp Val ThrGlu Thr Asn Gly Glu Asp Lys Met Phe 395 400 405 aag act ttc aac aca accaca gga ttg ccg gga agt cct caa gtt tca 1541 Lys Thr Phe Asn Thr Thr ThrGly Leu Pro Gly Ser Pro Gln Val Ser 410 415 420 aca ccg cct gca aac tcattc aat aaa ttt cct ccg tcg aca agt gat 1589 Thr Pro Pro Ala Asn Ser PheAsn Lys Phe Pro Pro Ser Thr Ser Asp 425 430 435 tcg cac aat tac ggt tccaga aca ggt aca cca atg tcc aat cac gtc 1637 Ser His Asn Tyr Gly Ser ArgThr Gly Thr Pro Met Ser Asn His Val 440 445 450 atg tct cca acc tta aataca gat tct agt tca gca aac ggg aaa ttc 1685 Met Ser Pro Thr Leu Asn ThrAsp Ser Ser Ser Ala Asn Gly Lys Phe 455 460 465 470 ata cca agt aga ccggct cct aag ccc cca tct tct gcg tcc gct tca 1733 Ile Pro Ser Arg Pro AlaPro Lys Pro Pro Ser Ser Ala Ser Ala Ser 475 480 485 gct cca att ata aaatca ccc gtc atg aat tct gcc gcc aat gtt tcg 1781 Ala Pro Ile Ile Lys SerPro Val Met Asn Ser Ala Ala Asn Val Ser 490 495 500 ccc ttg aag cag actcat gca cct aca act ccg aac agg acc agc cca 1829 Pro Leu Lys Gln Thr HisAla Pro Thr Thr Pro Asn Arg Thr Ser Pro 505 510 515 aac agg tcc tca atatca aga aat gcc act tta aaa aaa gag gag cag 1877 Asn Arg Ser Ser Ile SerArg Asn Ala Thr Leu Lys Lys Glu Glu Gln 520 525 530 cca cta cca cca atacct cca acc aaa tcc aaa acg tct cca atc atc 1925 Pro Leu Pro Pro Ile ProPro Thr Lys Ser Lys Thr Ser Pro Ile Ile 535 540 545 550 tcc aca gct cacaca cca cag caa gtt gct caa tcg cca aaa gcg ccg 1973 Ser Thr Ala His ThrPro Gln Gln Val Ala Gln Ser Pro Lys Ala Pro 555 560 565 gcg caa gag acggta acg aca cct act tcg aag cca gct caa gca aga 2021 Ala Gln Glu Thr ValThr Thr Pro Thr Ser Lys Pro Ala Gln Ala Arg 570 575 580 agc ttg tct aaagaa tta aat gag aaa aag aga gag gaa agg gaa aga 2069 Ser Leu Ser Lys GluLeu Asn Glu Lys Lys Arg Glu Glu Arg Glu Arg 585 590 595 cgt aaa aaa caacta tat gcc aaa ttg aac gaa att tgc tca gac ggt 2117 Arg Lys Lys Gln LeuTyr Ala Lys Leu Asn Glu Ile Cys Ser Asp Gly 600 605 610 gac cca agt acaaaa tat gcc aat tta gta aaa att ggt caa ggt gca 2165 Asp Pro Ser Thr LysTyr Ala Asn Leu Val Lys Ile Gly Gln Gly Ala 615 620 625 630 tca ggt ggtgtt tat act gct tat gaa ata ggt acg aat gtc tca gtg 2213 Ser Gly Gly ValTyr Thr Ala Tyr Glu Ile Gly Thr Asn Val Ser Val 635 640 645 gcc att aagcaa atg aat ctc gaa aag caa cca aaa aag gag cta atc 2261 Ala Ile Lys GlnMet Asn Leu Glu Lys Gln Pro Lys Lys Glu Leu Ile 650 655 660 atc aat gagatt ctg gtc atg aag ggt agc aaa cac cct aat ata gtt 2309 Ile Asn Glu IleLeu Val Met Lys Gly Ser Lys His Pro Asn Ile Val 665 670 675 aat ttc attgat tct tac gtt tta aaa ggc gac ctt tgg gtc att atg 2357 Asn Phe Ile AspSer Tyr Val Leu Lys Gly Asp Leu Trp Val Ile Met 680 685 690 gaa tac atggaa ggt ggc tcc tta act gat gtg gtc acc cat tgt att 2405 Glu Tyr Met GluGly Gly Ser Leu Thr Asp Val Val Thr His Cys Ile 695 700 705 710 ttg acagaa ggt caa att ggt gcc gtt tgt aga gaa act ttg agt ggg 2453 Leu Thr GluGly Gln Ile Gly Ala Val Cys Arg Glu Thr Leu Ser Gly 715 720 725 ttg gaattt tta cat tct aaa ggt gtt ctt cac aga gat atc aaa tcc 2501 Leu Glu PheLeu His Ser Lys Gly Val Leu His Arg Asp Ile Lys Ser 730 735 740 gat aacatc cta ttg tcc atg gaa ggg gat att aag tta acg gat ttc 2549 Asp Asn IleLeu Leu Ser Met Glu Gly Asp Ile Lys Leu Thr Asp Phe 745 750 755 ggt ttttgc gct caa atc aat gaa ttg aac ttg aaa aga act act atg 2597 Gly Phe CysAla Gln Ile Asn Glu Leu Asn Leu Lys Arg Thr Thr Met 760 765 770 gtg ggaacg cct tat tgg atg gcg cct gaa gtg gtt tct agg aaa gaa 2645 Val Gly ThrPro Tyr Trp Met Ala Pro Glu Val Val Ser Arg Lys Glu 775 780 785 790 tatggc cca aaa gta gat atc tgg tcg ttg ggt atc atg atc att gaa 2693 Tyr GlyPro Lys Val Asp Ile Trp Ser Leu Gly Ile Met Ile Ile Glu 795 800 805 atgatc gag ggg gag cct cca tat tta aat gaa acc ccg cta aga gca 2741 Met IleGlu Gly Glu Pro Pro Tyr Leu Asn Glu Thr Pro Leu Arg Ala 810 815 820 ctgtat tta att gct aca aat ggt aca ccc aag tta aag gaa ccc gag 2789 Leu TyrLeu Ile Ala Thr Asn Gly Thr Pro Lys Leu Lys Glu Pro Glu 825 830 835 aatcta tcg tca agc ttg aaa aaa ttc ctt gat tgg tgt tta tgt gtg 2837 Asn LeuSer Ser Ser Leu Lys Lys Phe Leu Asp Trp Cys Leu Cys Val 840 845 850 gagccc gaa gat aga gca agc gct acg gaa ttg ctt cat gat gaa tat 2885 Glu ProGlu Asp Arg Ala Ser Ala Thr Glu Leu Leu His Asp Glu Tyr 855 860 865 870atc acg gag ata gct gaa gcc aat tcc tca ttg gcc ccg cta gtc aag 2933 IleThr Glu Ile Ala Glu Ala Asn Ser Ser Leu Ala Pro Leu Val Lys 875 880 885tta gca aga ttg aag aaa gta gct gag aac atg gat gct gat gaa gat 2981 LeuAla Arg Leu Lys Lys Val Ala Glu Asn Met Asp Ala Asp Glu Asp 890 895 900aat gac gac gat aac gac aac gag cat att aat aag aca aac aat tgt 3029 AsnAsp Asp Asp Asn Asp Asn Glu His Ile Asn Lys Thr Asn Asn Cys 905 910 915gac gac aat aac gat agc aaa gaa acc gta aat ttg gac gta act gaa 3077 AspAsp Asn Asn Asp Ser Lys Glu Thr Val Asn Leu Asp Val Thr Glu 920 925 930gat gat aaa caa aag taaacgtagc aagcagggta caccttatta tcgacaaagt 3132 AspAsp Lys Gln Lys 935 atatacacag ttgtgactgg cataaaaatt cttttcatatatcttatcgt gtatatttgg 3192 acattttata acacatccca ctctaattca caacttcattaacgaaattt aaataaatca 3252 cgacaacagt tttgcttaaa actgaggaat attgaaaccaactcaaattc ttcctaattt 3312 caggcgtata aaaataacaa attctcatcg attgtcgggtaccattacac gaacatctgt 3372 ctgcgttcta tgtaacgaag gagaggtatt atccaattttggaaatatcc gtaatattgt 3432 ccttagtgca cgaactatat tatcccgcaa attcagggaaaagaaaagaa gtagaaaaaa 3492 aaaaatacca tgggagtcag ttcttgttca gctgagagaattacgcttgt ttcttatttc 3552 ccacatatac gagaaattcc taccgatata acatcctctctcgtcttcta gaattttcca 3612 gttgagtgaa gttttttatt ttcataaact aacaagattatttcatggaa cagtgacgga 3672 aaggattttc taaaggcatt gttagaaaaa atggttgacgactcaaacta tcttacacca 3732 catgaaactg cattagcggt ggtggccact gcaatgaagaaagcaagact gcaactagat 3792 acattgctaa taaattccat acttggtggc gttctgtttagtagtggttc gttctattgg 3852 tagcggtata ttccgaagat cctgacatag tcgcacgaaacccgggtatt gtgaatctta 3912 ttactggtgt taatttcgcc atgggactat tctatgtagtaatgatgggt gctgacctct 3972 tcaactctaa tatcctattt ttctccgttg gagttctgagaaaagcagta actatctatg 4032 atttgatgat ttcgtgggtt gtcagttggt taggtaatattgctggctca ctttttgttt 4092 catatctttt tggtcatctt tctggtatta gttctcagaagctt 4136 29 939 PRT Saccharomyces cerevisiae 29 Met Ser Asn Asp Pro SerAla Val Ser Glu Leu Pro Asp Lys Asp Ser 1 5 10 15 Leu Asp Asn Gly IleSer Asn Asp Asn Glu Arg Ala Met Gly Gly Asn 20 25 30 Gly Asp Gly Gly AspGly Leu Arg Leu Pro Arg Thr Thr Gly Thr Leu 35 40 45 Asn Val Asn Ala LeuGln Lys Gly Thr Asn Ala Ala His Glu Ala Gly 50 55 60 Gly Tyr Lys Ser MetAsp Pro Ala Lys Asn Ala Glu Thr Thr Asn Asp 65 70 75 80 Asp Asp Asn AsnVal Val Ser Leu Asp Asp Pro Ile Gln Phe Thr Arg 85 90 95 Val Ser Ser SerSer Val Ile Ser Gly Met Ser Ser Ser Met Ser Pro 100 105 110 His Ser AsnIle Asp Glu Thr Lys Ser Leu Glu Ala Val Thr Pro Asn 115 120 125 Ile AsnThr Ser Asn Ile Thr Pro Asp His Ser Ala Asp Asn Thr Phe 130 135 140 SerThr Ile Asn Ala Ser Glu Ser Asp His Gln Phe Asn Asp Thr Leu 145 150 155160 Leu Ser Lys Leu Ser Leu Thr Asp Ser Thr Glu Thr Ile Glu Asn Asn 165170 175 Ala Thr Val Lys His Gln Gln Pro Val Ala Ser Ser Thr Val Asn Ser180 185 190 Asn Lys Ser Ser Thr Asp Ile Arg Arg Ala Thr Pro Val Ser ThrPro 195 200 205 Val Ile Ser Lys Pro Ser Met Thr Thr Thr Pro Arg Gln IleAsn Ser 210 215 220 Ala Ser His Ser Leu Ser Asn Pro Lys His Lys Gln HisLys Pro Lys 225 230 235 240 Val Lys Pro Ser Lys Pro Glu Ala Lys Ser LysPro Val Ser Val Lys 245 250 255 Lys Ser Phe Pro Ser Lys Asn Pro Leu LysAsn Ser Ser Pro Pro Lys 260 265 270 Lys Gln Thr Glu Lys Ser Tyr Tyr SerSer Ser Ser Lys Lys Arg Lys 275 280 285 Ser Gly Ser Asn Ser Gly Thr LeuArg Met Lys Asp Val Phe Thr Ser 290 295 300 Phe Val Gln Asn Ile Lys ArgAsn Ser Gln Asp Asp Lys Arg Ala Ser 305 310 315 320 Ser Ser Ser Asn AsnSer Ser Ser Ser Ser Ile Thr Thr Ala Leu Arg 325 330 335 Ile Ser Thr ProTyr Asn Ala Lys His Ile His His Val Gly Val Asp 340 345 350 Ser Lys ThrGly Glu Tyr Thr Gly Leu Pro Glu Glu Trp Glu Lys Leu 355 360 365 Leu ThrSer Ser Gly Ile Ser Lys Arg Glu Gln Gln Gln Asn Met Gln 370 375 380 AlaVal Met Asp Ile Val Lys Phe Tyr Gln Asp Val Thr Glu Thr Asn 385 390 395400 Gly Glu Asp Lys Met Phe Lys Thr Phe Asn Thr Thr Thr Gly Leu Pro 405410 415 Gly Ser Pro Gln Val Ser Thr Pro Pro Ala Asn Ser Phe Asn Lys Phe420 425 430 Pro Pro Ser Thr Ser Asp Ser His Asn Tyr Gly Ser Arg Thr GlyThr 435 440 445 Pro Met Ser Asn His Val Met Ser Pro Thr Leu Asn Thr AspSer Ser 450 455 460 Ser Ala Asn Gly Lys Phe Ile Pro Ser Arg Pro Ala ProLys Pro Pro 465 470 475 480 Ser Ser Ala Ser Ala Ser Ala Pro Ile Ile LysSer Pro Val Met Asn 485 490 495 Ser Ala Ala Asn Val Ser Pro Leu Lys GlnThr His Ala Pro Thr Thr 500 505 510 Pro Asn Arg Thr Ser Pro Asn Arg SerSer Ile Ser Arg Asn Ala Thr 515 520 525 Leu Lys Lys Glu Glu Gln Pro LeuPro Pro Ile Pro Pro Thr Lys Ser 530 535 540 Lys Thr Ser Pro Ile Ile SerThr Ala His Thr Pro Gln Gln Val Ala 545 550 555 560 Gln Ser Pro Lys AlaPro Ala Gln Glu Thr Val Thr Thr Pro Thr Ser 565 570 575 Lys Pro Ala GlnAla Arg Ser Leu Ser Lys Glu Leu Asn Glu Lys Lys 580 585 590 Arg Glu GluArg Glu Arg Arg Lys Lys Gln Leu Tyr Ala Lys Leu Asn 595 600 605 Glu IleCys Ser Asp Gly Asp Pro Ser Thr Lys Tyr Ala Asn Leu Val 610 615 620 LysIle Gly Gln Gly Ala Ser Gly Gly Val Tyr Thr Ala Tyr Glu Ile 625 630 635640 Gly Thr Asn Val Ser Val Ala Ile Lys Gln Met Asn Leu Glu Lys Gln 645650 655 Pro Lys Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Gly Ser660 665 670 Lys His Pro Asn Ile Val Asn Phe Ile Asp Ser Tyr Val Leu LysGly 675 680 685 Asp Leu Trp Val Ile Met Glu Tyr Met Glu Gly Gly Ser LeuThr Asp 690 695 700 Val Val Thr His Cys Ile Leu Thr Glu Gly Gln Ile GlyAla Val Cys 705 710 715 720 Arg Glu Thr Leu Ser Gly Leu Glu Phe Leu HisSer Lys Gly Val Leu 725 730 735 His Arg Asp Ile Lys Ser Asp Asn Ile LeuLeu Ser Met Glu Gly Asp 740 745 750 Ile Lys Leu Thr Asp Phe Gly Phe CysAla Gln Ile Asn Glu Leu Asn 755 760 765 Leu Lys Arg Thr Thr Met Val GlyThr Pro Tyr Trp Met Ala Pro Glu 770 775 780 Val Val Ser Arg Lys Glu TyrGly Pro Lys Val Asp Ile Trp Ser Leu 785 790 795 800 Gly Ile Met Ile IleGlu Met Ile Glu Gly Glu Pro Pro Tyr Leu Asn 805 810 815 Glu Thr Pro LeuArg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro 820 825 830 Lys Leu LysGlu Pro Glu Asn Leu Ser Ser Ser Leu Lys Lys Phe Leu 835 840 845 Asp TrpCys Leu Cys Val Glu Pro Glu Asp Arg Ala Ser Ala Thr Glu 850 855 860 LeuLeu His Asp Glu Tyr Ile Thr Glu Ile Ala Glu Ala Asn Ser Ser 865 870 875880 Leu Ala Pro Leu Val Lys Leu Ala Arg Leu Lys Lys Val Ala Glu Asn 885890 895 Met Asp Ala Asp Glu Asp Asn Asp Asp Asp Asn Asp Asn Glu His Ile900 905 910 Asn Lys Thr Asn Asn Cys Asp Asp Asn Asn Asp Ser Lys Glu ThrVal 915 920 925 Asn Leu Asp Val Thr Glu Asp Asp Lys Gln Lys 930 935 302325 DNA Saccharomyces cerevisiae CDS (496)..(1764) 30 gaaaaatgtttcaggaagag atactgcgta aaaaaaagac acatgtgtta cgcaggaaaa 60 agtttgtgaggctttttgcc ttaacagatt gacttgtagc cctgttaggt ttacccaaca 120 tttgtttttctgtgtgtcga aaattttttc agagtgtttt caactgacac ttgcctgttt 180 catattagttgtaacttaaa ctttcaaaca taaaactttt ttggaagtcc atccttcaca 240 tgacttgaatcccttcaata tcgaaacagt tatcctcaaa atctcttatc acttttctaa 300 ttgttttcttcccctttttt gtagtaactc gctgtaaagc acattttatt cataatctcc 360 tttgtgccagaactcaaggt caataggcca gaattattgg aaggaaagag ggaagaaaat 420 acgatattgctagttcatta agtcaaggaa gaaaatactc aaaaaactgt acagctcaat 480 caggtacacattacg atg gca gca cat cag atg gac tcg ata acg tat tct 531 Met Ala AlaHis Gln Met Asp Ser Ile Thr Tyr Ser 1 5 10 aat aat gtc acc caa cag tatata caa cca caa agt cta cag gat atc 579 Asn Asn Val Thr Gln Gln Tyr IleGln Pro Gln Ser Leu Gln Asp Ile 15 20 25 tct gca gtg gag gaa gaa att caaaat aaa ata gag gcc gcc aga caa 627 Ser Ala Val Glu Glu Glu Ile Gln AsnLys Ile Glu Ala Ala Arg Gln 30 35 40 gag agt aaa cag ctt cat gct caa ataaat aaa gca aaa cac aag ata 675 Glu Ser Lys Gln Leu His Ala Gln Ile AsnLys Ala Lys His Lys Ile 45 50 55 60 caa gat gca agc tta ttc cag atg gccaac aaa gtt act tcg ttg acc 723 Gln Asp Ala Ser Leu Phe Gln Met Ala AsnLys Val Thr Ser Leu Thr 65 70 75 aaa aat aag atc aac tta aag cca aat atcgtg ttg aaa ggc cat aat 771 Lys Asn Lys Ile Asn Leu Lys Pro Asn Ile ValLeu Lys Gly His Asn 80 85 90 aat aaa atc tca gat ttt cgg tgg agt cga gattca aaa cgt att ttg 819 Asn Lys Ile Ser Asp Phe Arg Trp Ser Arg Asp SerLys Arg Ile Leu 95 100 105 agt gca agt caa gat ggc ttt atg ctt ata tgggac agt gct tca ggt 867 Ser Ala Ser Gln Asp Gly Phe Met Leu Ile Trp AspSer Ala Ser Gly 110 115 120 tta aaa cag aac gct att cca tta gat tct caatgg gtt ctt tcc tgc 915 Leu Lys Gln Asn Ala Ile Pro Leu Asp Ser Gln TrpVal Leu Ser Cys 125 130 135 140 gct att tcg cca tcg agt act ttg gta gcaagc gca gga tta aac aat 963 Ala Ile Ser Pro Ser Ser Thr Leu Val Ala SerAla Gly Leu Asn Asn 145 150 155 aac tgt acc att tat aga gtt tcg aaa gaaaac aga gta gcg caa aac 1011 Asn Cys Thr Ile Tyr Arg Val Ser Lys Glu AsnArg Val Ala Gln Asn 160 165 170 gtt gcg tca att ttc aaa gga cat act tgctat att tct gac att gaa 1059 Val Ala Ser Ile Phe Lys Gly His Thr Cys TyrIle Ser Asp Ile Glu 175 180 185 ttt aca gat aac gca cat ata ttg aca gcaagt ggg gat atg aca tgt 1107 Phe Thr Asp Asn Ala His Ile Leu Thr Ala SerGly Asp Met Thr Cys 190 195 200 gcc ttg tgg gat ata ccg aaa gca aag agggtg aga gaa tat tct gac 1155 Ala Leu Trp Asp Ile Pro Lys Ala Lys Arg ValArg Glu Tyr Ser Asp 205 210 215 220 cat tta ggt gat gtt ttg gca tta gctatt cct gaa gag cca aac tta 1203 His Leu Gly Asp Val Leu Ala Leu Ala IlePro Glu Glu Pro Asn Leu 225 230 235 gaa aat tct tcg aac aca ttc gct agctgt gga tca gac ggg tat act 1251 Glu Asn Ser Ser Asn Thr Phe Ala Ser CysGly Ser Asp Gly Tyr Thr 240 245 250 tac ata tgg gat agc aga tct ccg tccgct gta caa agc ttt tac gtt 1299 Tyr Ile Trp Asp Ser Arg Ser Pro Ser AlaVal Gln Ser Phe Tyr Val 255 260 265 aac gat agt gat att aat gca ctt cgtttt ttc aaa gac ggg atg tcg 1347 Asn Asp Ser Asp Ile Asn Ala Leu Arg PhePhe Lys Asp Gly Met Ser 270 275 280 att gtt gca gga agt gac aat ggt gcgata aat atg tat gat tta agg 1395 Ile Val Ala Gly Ser Asp Asn Gly Ala IleAsn Met Tyr Asp Leu Arg 285 290 295 300 tcg gac tgt tct att gct act ttttct ctt ttt cga ggt tat gaa gaa 1443 Ser Asp Cys Ser Ile Ala Thr Phe SerLeu Phe Arg Gly Tyr Glu Glu 305 310 315 cgt acc cct acc cct act tat atggca gct aac atg gag tac aat acc 1491 Arg Thr Pro Thr Pro Thr Tyr Met AlaAla Asn Met Glu Tyr Asn Thr 320 325 330 gcg caa tcg cca caa act tta aaatca aca agc tca agc tat cta gac 1539 Ala Gln Ser Pro Gln Thr Leu Lys SerThr Ser Ser Ser Tyr Leu Asp 335 340 345 aac caa ggc gtt gtt tct tta gatttt agt gca tct gga aga ttg atg 1587 Asn Gln Gly Val Val Ser Leu Asp PheSer Ala Ser Gly Arg Leu Met 350 355 360 tac tca tgc tat aca gac att ggttgt gtt gtg tgg gat gta tta aaa 1635 Tyr Ser Cys Tyr Thr Asp Ile Gly CysVal Val Trp Asp Val Leu Lys 365 370 375 380 gga gag att gtt gga aaa ttagaa ggt cat ggt ggc aga gtc act ggt 1683 Gly Glu Ile Val Gly Lys Leu GluGly His Gly Gly Arg Val Thr Gly 385 390 395 gtg cgc tcg agt cca gat gggtta gct gta tgt aca ggt tca tgg gac 1731 Val Arg Ser Ser Pro Asp Gly LeuAla Val Cys Thr Gly Ser Trp Asp 400 405 410 tca acc atg aaa ata tgg tctcca ggt tat caa tagcttcgaa ttggaaatac 1784 Ser Thr Met Lys Ile Trp SerPro Gly Tyr Gln 415 420 tgtgagcagt aattatcaat ggatgctatt atataaatatacatacctac acccatccca 1844 tatttacata gaataacaac agtaacatta gttctgtggaagcgcaaaaa cgtcctttaa 1904 taaagtaagt caaaacattc aacaatgaaa attcaaagcattgtcatttg cttccttttt 1964 ctctttggga taaacgaaac aaaaacgaac aaaatgtcatgcactcaaaa attcttttca 2024 atcgttttgg aaacagtatt attcactgac ttatttgaccaacttgctag aatcatctat 2084 gttttcaggc attgtttaat ttcatgatgg ctgtccctactttagcttgt tatgagcctt 2144 cactggctcg tccttatgta ttgcgtctga cccaaaatttgtcctttctt gtttagtgga 2204 atttttgttc ggtaatttca aaaatgctga attttcattaacaaatcatc tggtagttgt 2264 gttataaaca taaaaaactg ctcccttctg ggatgattttcaattgctct ctgtactgca 2324 g 2325 31 423 PRT Saccharomyces cerevisiae 31Met Ala Ala His Gln Met Asp Ser Ile Thr Tyr Ser Asn Asn Val Thr 1 5 1015 Gln Gln Tyr Ile Gln Pro Gln Ser Leu Gln Asp Ile Ser Ala Val Glu 20 2530 Glu Glu Ile Gln Asn Lys Ile Glu Ala Ala Arg Gln Glu Ser Lys Gln 35 4045 Leu His Ala Gln Ile Asn Lys Ala Lys His Lys Ile Gln Asp Ala Ser 50 5560 Leu Phe Gln Met Ala Asn Lys Val Thr Ser Leu Thr Lys Asn Lys Ile 65 7075 80 Asn Leu Lys Pro Asn Ile Val Leu Lys Gly His Asn Asn Lys Ile Ser 8590 95 Asp Phe Arg Trp Ser Arg Asp Ser Lys Arg Ile Leu Ser Ala Ser Gln100 105 110 Asp Gly Phe Met Leu Ile Trp Asp Ser Ala Ser Gly Leu Lys GlnAsn 115 120 125 Ala Ile Pro Leu Asp Ser Gln Trp Val Leu Ser Cys Ala IleSer Pro 130 135 140 Ser Ser Thr Leu Val Ala Ser Ala Gly Leu Asn Asn AsnCys Thr Ile 145 150 155 160 Tyr Arg Val Ser Lys Glu Asn Arg Val Ala GlnAsn Val Ala Ser Ile 165 170 175 Phe Lys Gly His Thr Cys Tyr Ile Ser AspIle Glu Phe Thr Asp Asn 180 185 190 Ala His Ile Leu Thr Ala Ser Gly AspMet Thr Cys Ala Leu Trp Asp 195 200 205 Ile Pro Lys Ala Lys Arg Val ArgGlu Tyr Ser Asp His Leu Gly Asp 210 215 220 Val Leu Ala Leu Ala Ile ProGlu Glu Pro Asn Leu Glu Asn Ser Ser 225 230 235 240 Asn Thr Phe Ala SerCys Gly Ser Asp Gly Tyr Thr Tyr Ile Trp Asp 245 250 255 Ser Arg Ser ProSer Ala Val Gln Ser Phe Tyr Val Asn Asp Ser Asp 260 265 270 Ile Asn AlaLeu Arg Phe Phe Lys Asp Gly Met Ser Ile Val Ala Gly 275 280 285 Ser AspAsn Gly Ala Ile Asn Met Tyr Asp Leu Arg Ser Asp Cys Ser 290 295 300 IleAla Thr Phe Ser Leu Phe Arg Gly Tyr Glu Glu Arg Thr Pro Thr 305 310 315320 Pro Thr Tyr Met Ala Ala Asn Met Glu Tyr Asn Thr Ala Gln Ser Pro 325330 335 Gln Thr Leu Lys Ser Thr Ser Ser Ser Tyr Leu Asp Asn Gln Gly Val340 345 350 Val Ser Leu Asp Phe Ser Ala Ser Gly Arg Leu Met Tyr Ser CysTyr 355 360 365 Thr Asp Ile Gly Cys Val Val Trp Asp Val Leu Lys Gly GluIle Val 370 375 380 Gly Lys Leu Glu Gly His Gly Gly Arg Val Thr Gly ValArg Ser Ser 385 390 395 400 Pro Asp Gly Leu Ala Val Cys Thr Gly Ser TrpAsp Ser Thr Met Lys 405 410 415 Ile Trp Ser Pro Gly Tyr Gln 420

What is claimed is:
 1. An isolated Ste4p/G_(β)-binding polypeptide orfragment thereof wherein said isolated Ste4p/G_(β)-binding polypeptideis a Ste20p/PAK polypeptide which directly binds to a Ste4p/G_(β)polypeptide or fragment thereof.
 2. The isolated Ste4p/G_(β)-bindingpolypeptide of claim 1, comprising an amino acid sequence having atleast 95% identity to the amino acid sequence selected from the groupconsisting of: a) a full length amino acid sequence of SEQ. ID. NO.:29;b) an amino acid sequence having amino acids 495 to 939 of SEQ. ID.NO.:29; c) an amino acid sequence having amino acids 495 to 888 of SEQ.ID. NO.:29; d) an amino acid sequence having amino acids 819 to 939 ofSEQ. ID. NO.:29; e) an amino acid sequence having amino acids 819 to 892of SEQ. ID. NO.:29; f) an amino acid sequence having amino acids 876 to939 of SEQ. ID. NO.:29; and g) an amino acid sequence having amino acids876 to 892 of SEQ. ID. NO.:29.
 3. The isolated Ste4p/G_(β)-bindingpolypeptide of claim 1, comprising an amino acid sequence having atleast 95% identity to the amino acid sequence selected from the groupconsisting of: a) an amino acid sequence of SEQ. ID. NO.:29; b) an aminoacid sequence of SEQ. ID. NO.: 1; c) an amino acid sequence of SEQ. ID.NO.:2; d) an amino acid sequence of SEQ. ID. NO.:3; e) an amino acidsequence of SEQ. ID. NO.:4; e) an amino acid sequence of SEQ. ID. NO.:5;g) an amino acid sequence of SEQ. ID. NO.:6; h) an amino acid sequenceof SEQ. ID. NO.:7; i) an amino acid sequence of SEQ. ID. NO.:8; j) anamino acid sequence of SEQ. ID. NO.:9; k) an amino acid sequence of SEQ.ID. NO.:10 l) an amino acid sequence of SEQ. ID. NO.:11; m) an aminoacid sequence of SEQ. ID. NO.:12; and n) an amino acid sequence of SEQ.ID. NO.:13.
 4. The isolated Ste4p/G_(β)binding polypeptide of claim 1,joined to a heterologous polypeptide, thereby forming an isolatedchimeric polypeptide which directly binds to a Ste4p/G_(β) polypeptideor fragment thereof.
 5. The isolated Ste4p/G_(β)-binding polypeptide ofclaim 4, comprising amino acid sequence SSLφPLI_(V)Xφφβ, wherein φ isselected from A, I, L, M, S, T, and β is selected from H, K, and R. 6.The isolated Ste4p/G_(β)-binding polypeptide of claim 5, wherein saidheterologous polypeptide is Glutathione-S-transferase.
 7. An isolatednucleic acid molecule comprising a nucleic acid sequence which encodes aSte4p/G_(β) binding domain of Ste20p/PAK.
 8. The isolated nucleic acidof claim 7, comprising a nucleic acid sequence at least 90% identical toa sequence selected from the group consisting of: a) a nucleotidesequence encoding a full length amino acid sequence of SEQ. ID. NO.:29;b) a nucleotide sequence encoding an amino acid sequence having aminoacid 495 to 939 of SEQ. ID. NO.:29; c) a nucleotide sequence encoding anamino acid sequence having amino acid 495 to 888 of SEQ. ID. NO.:29; d)a nucleotide sequence encoding an amino acid sequence having amino acid819 to 939 of SEQ. ID. NO.:29; e) a nucleotide sequence encoding anamino acid sequence having amino acid 819 to 892 of SEQ. ID. NO.:29; f)a nucleotide sequence encoding an amino acid sequence having amino acid876 to 939 of SEQ. ID. NO.:29; g) a nucleotide sequence encoding anamino acid sequence having amino acid 876 to 892 of SEQ. ID. NO.:29; andh) a nucleotide sequence which hybridizes to a)-g) under high stringencyconditions.
 9. The isolated nucleic acid of claim 7, comprising anucleic acid sequence at least 90% identical to a sequence selected fromthe group consisting of: a) a nucleotide sequence encoding an amino acidsequence of SEQ. ID. NO.:29; b) a nucleotide sequence encoding an aminoacid sequence of SEQ. ID. NO.:1; c) a nucleotide sequence encoding anamino acid sequence of SEQ. ID. NO.:2; d) a nucleotide sequence encodingan amino acid sequence of SEQ. ID. NO.:3; e) a nucleotide sequenceencoding an amino acid sequence of SEQ. ID. NO.:4; f) a nucleotidesequence encoding an amino acid sequence of SEQ. ID. NO.:5; g) anucleotide sequence encoding an amino acid sequence of SEQ. ID. NO.:6;h) a nucleotide sequence encoding an amino acid sequence of SEQ. ID.NO.:7; i) a nucleotide sequence encoding an amino acid sequence of SEQ.ID. NO.:8; j) a nucleotide sequence encoding an amino acid sequence ofSEQ. ID. NO.:9; k) a nucleotide sequence encoding an amino acid sequenceof SEQ. ID. NO.:10; l) a nucleotide sequence encoding an amino acidsequence of SEQ. ID. NO.: 11; m) a nucleotide sequence encoding an aminoacid sequence of SEQ. ID. NO.:12; and n) a nucleotide sequence encodingan amino acid sequence of SEQ. ID. NO.:13; o) a nucleotide sequenceencoding an amino acid sequence SSLφPLI_(V)Xφφβ, wherein φ is selectedfrom A, I, L, M, S, T, and β is selected from H, K, and R; p) anucleotide sequence which hybridizes to a)-e) under high stringencyconditions.
 10. An isolated nucleic acid molecule encoding a Ste4p/G_(β)binding domain containing a fusion protein, said Ste4p/G_(β) interactiondomain being fused to a heterologous polypeptide sequence, wherein saidSte4p/G_(β) binding domain is encoded by the nucleic acid molecule ofclaim
 7. 11. The isolated nucleic acid molecule of claim 10 wherein saidSte4p/G_(β) binding domain comprises amino acid sequenceSSLφPLI_(V)Xφφβ, wherein φ is selected from A, I, L, M, S, T, and β isselected from H, K, and R.
 12. The isolated nucleic acid molecule ofclaim 11, wherein said heterologous polypeptide sequence encodesGlutathione-S-transferase.
 13. An isolated nucleic acid moleculecomprising a nucleic acid sequence which encodes a Ste20p/PAK bindingdomain of Ste4p/G_(β), wherein said Ste 20p/PAK binding domain is aSte4p/G_(β) polypeptide which directly binds to a Ste20p/PAK polypeptideor fragment thereof.
 14. The isolated nucleic acid molecule of claim 13,wherein said sequence encodes a polypeptide comprising an amino acidsequence having at least 95% identity to the amino acid sequenceselected from the group consisting of: a) a full length amino acidsequence of SEQ. ID. NO.:21; b) an amino acid sequence having aminoacids 1 to 150 of SEQ. ID. NO.:21; c) an amino acid sequence havingamino acids 1 to 100 of SEQ. ID. NO.:21; d) an amino acid sequencehaving amino acids 1 to 80 of SEQ. ID. NO.:21; e) an amino acid sequenceof SEQ. ID. NO.:22; f) an amino acid sequence of SEQ. ID. NO.:23; g) anamino acid sequence of SEQ. ID. NO.:24; h) an amino acid sequence ofSEQ. ID. NO.:25; and i) an amino acid sequence of SEQ. ID. NO.:26.
 15. Avector comprising the nucleic acid molecule of claim
 7. 16. A host cellharboring the nucleic acid molecule of claim
 15. 17. The host cell ofclaim 16 further harboring a vector comprising a nucleic acid moleculewhich comprises a nucleic acid sequence encoding a Ste20p/PAKinteraction domain of Ste4p/G_(β).
 18. The host cell of claim 17,wherein at least one assayable metabolic function is dependent on theinteraction of said nucleic acid sequences encoding the Ste4p/G_(β)interaction domain of Ste20p/PAK and the Ste20p/PAK interaction domainof Ste4p/G_(β).
 19. A method of assaying compounds having the ability tomodulate the interaction between Ste20p/PAK and Ste4p/G_(β) comprisingthe steps of: a) incubating the host cell of claim 18 with a testcompound; b) assaying said at least one metabolic function dependent onsaid interaction between Ste20p/PAK and Ste4p/G_(β); and c) identifyingsaid compound as a modulator of said interaction.
 20. A method ofassaying compounds having the ability to modulate the interactionbetween Ste20p/PAK and Ste4p/G_(β) comprising the steps of: a)incubating the isolated Ste4p/G_(β) polypeptide of claim 5 with anisolated Ste20p/PAK-binding polypeptide or fragment thereof, whereinsaid isolated Ste20p/PAK-binding polypeptide is a Ste4p/G_(β)polypeptide which directly binds to a Ste20p/PAK polypeptide or fragmentthereof, in the presence of a test compound; b) assaying in vitro thebinding between said isolated Ste4p/G_(β) polypeptide and said isolatedSte20p/PAK polypeptide; and c) identifying said compound as a modulatorof said interaction.